Postnatal and adult exposure to estradiol differentially influences adult neurogenesis in the main and accessory olfactory bulb of female mice

Abstract

Neurons incorporated into the adult main olfactory bulb (MOB) and accessory olfactory bulb (AOB) derive from the subventricular zone (SVZ). Despite some recent studies on the role of olfactory neurogenesis in sociosexual behaviors mediated by hormones, data on the implication of estrogens are still lacking. Taking advantage of female aromatase-knockout (ArKO) mice, which are unable to produce estradiol across their life span, we investigated the role of estradiol exposure during early postnatal and adult periods on adult neurogenesis in the MOB and AOB. We found that proliferation of progenitor cells in the adult female SVZ was not influenced by estradiol. However, whereas adult exposure to estradiol influences the turnover of MOB newborn neurons, the survival of those in the AOB depends on exposure to estradiol during the early postnatal period. Finally, based on their expression of Zif268, we showed that newborn neurons in the MOB responded to sociosexual odors, albeit to a lesser extent in ArKO females, suggesting a contribution of estradiol during the early postnatal period to this response. Together, these results suggest that the survival and functional integration of newborn neurons in the adult female MOB and AOB are differentially influenced by estrogens from the early postnatal period to adulthood.

Figure 1.

Effect of estradiol on cell proliferation in the SVZ. A) Adult WT and ArKO female mice were given 3 different hormonal treatments for 3 wk: ovariectomy (OVX), ovary-intact estrous cycle (intact), and ovariectomy + implantation of an estradiol capsule (OVX-E2). Mice were injected with BrdU (2 injections at 2 h interval) on d 21 and sacrificed (S) 2 h afterward for assessment of cell proliferation in the SVZ. Stage of the estrous cycle of intact mice was determined before the first BrdU injection by analysis of vaginal smears (star). B) Representative image showing BrdU⁺ cells in the SVZ, analyzed 2 h after BrdU injections. LV, lateral ventricle. Scale bar = 50 μm. C) Total number of BrdU⁺ cells in the SVZ of the 6 experimental groups. D) Volume of the SVZ in the experimental groups. OVX: WT, n = 6; ArKO, n = 6. Intact: WT, n = 6; ArKO, n = 4. OVX-E2: WT, n = 5; ArKO, n = 4. Values are expressed as means ± se.

Figure 2.

Experimental design for assessing cell survival and the activation of newborn neurons in the MOB and AOB. Three additional groups of adult WT and ArKO female mice were submitted to 3 distinct hormonal treatments for 3 wk. OVX and OVX-E2 groups were ovariectomized on the first day and returned to their home cages for 3 wk. On d 21, all mice received 4 BrdU injections (at 2-h intervals) and were returned to their respective cages for 4 wk. OVX-E2 mice received implants the day after BrdU injections and kept the implants for the remaining 4 wk of the experiment. Vaginal smears (stars) were performed in intact mice at the time of the first BrdU injection on d 21 and again on the final day of the experiment, just before sacrifice (S). On the final day, all mice were separated into 2 subgroups, isolated for 2 h before stimulation, and exposed for 30 min to either deionized water or to male urinary odors. Mice were killed 60 min later to assess immediate Zif268 gene expression.

Figure 3.

Influence of estradiol on the survival of newborn neurons in the adult MOB. A) Representative images showing newly generated BrdU cells in the GrCL of the MOB, analyzed 4 wk after BrdU injections. Number of BrdU cells was decreased in WT-OVX-E2 and ArKO-OVX-E2 compared with WT-OVX and ArKO-OVX female mice. SEL, subependymal layer; Mi, mitral cell layer. B) Total number of BrdU⁺ cells in the MOB GrCL from the 6 experimental groups. C) Representative image of BrdU-NeuN double-labeled cell (arrow) under confocal microscopy. D) Percentage of BrdU-NeuN double-labeled cells counted in the GrCL of the MOB. OVX: WT, n = 6; ArKO, n = 6. Intact: WT, n = 4; ArKO, n = 4. OVX-E2: WT, n = 6; ArKO, n = 5. Values are expressed as means ± se. Scale bars = 60 μm (A); 6 μm (C). *P < 0.05, ***P < 0.005 vs. OVX; ^SP < 0.05 vs. WT; 2-way ANOVA followed by Fisher's post hoc test.

Figure 4.

Influence of estradiol on the survival of newborn neurons in the adult AOB. A) Representative images showing newly generated BrdU cells in the GrCL of the AOB, analyzed 4 wk after BrdU injections. Number of BrdU cells was decreased in ArKO-OVX compared with WT-OVX, WT-OVX-E2, or ArKO-OVX-E2 mice. LOT, lateral olfactory tract. B) Total number of BrdU⁺ cells in the AOB GrCL. C) Representative image of a BrdU-NeuN double-labeled cell (arrow) under confocal microscopy. D) Percentage of BrdU-NeuN double-labeled cells counted in the GrCL of the AOB. OVX: WT, n = 6; ArKO, n = 5. Intact: WT, n = 5; ArKO, n = 4. OVX-E2: WT, n = 5; ArKO, n = 5. Values are means ± se. Scale bars = 50 μm (A); 6 μm (C). *P < 0.05 vs. OVX; ^SP < 0.05, ^SSSP < 0.005 vs. WT; 2-way ANOVA followed by Fisher's post hoc test.

Figure 5.

Coexpression of BrdU and Zif268 in the MOB and AOB granular cell layers after exposure to male urinary odors. A) Representative image of a BrdU⁺ Zif268⁺ double-labeled cell (asterisk), a BrdU⁺ Zif268⁻ cell (arrow), and a Zif268⁺ BrdU⁻ cell (arrowhead) in the GrCL of the MOB after stimulation to male urinary odors. B) High magnification showing a BrdU⁺ Zif268⁺ double-labeled cell (asterisk), a BrdU⁺ Zif268⁻ cell (arrow) and a Zif268⁺ BrdU⁻ cell (arrowhead) in the GrCL of the AOB after stimulation to male urinary odors. C) Percentage of BrdU-Zif268 cells after exposure to water (control) or to male urinary odors counted in the GrCL of the MOB. D) Percentage of BrdU-Zif268 cells after exposure to water (control) or to male urinary odors counted in the GrCL of the AOB. OVX: WT, n = 6; ArKO, n = 6. Intact: WT, n = 6; ArKO, n = 4. OVX-E2: WT, n = 6; ArKO, n = 6. Values are means ± se. Scale bars = 6 μm. *P < 0.05, ***P < 0.005 vs. control; ^SP < 0.05, ^SSSP < 0.005 vs. WT; 2-way ANOVA followed by Fisher's post hoc test.