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. Author manuscript; available in PMC: 2016 May 5.
Published in final edited form as: Mol Cell Endocrinol. 2015 Feb 17;406:19–26. doi: 10.1016/j.mce.2015.02.013

Expression of Estrogen Receptor α in the Mouse Cerebral Cortex

Alicia K Dietrich 1, Gwendolyn I Humphreys 1, Ann M Nardulli 1
PMCID: PMC4773199  NIHMSID: NIHMS665132  PMID: 25700604

Abstract

Although estrogen receptor alpha (ERα) and 17β-estradiol play critical roles in protecting the cerebral cortex from ischemia-induced damage, there has been some controversy about the expression of ERα in this region of the brain. We have examined ERα mRNA and protein levels in the cerebral cortices of female mice at postnatal days 5 and 17 and at 4, 13, and 18 months of age. We found that although ERα transcript levels declined from postnatal day 5 through 18 months of age, ERα protein levels remained stable. Importantly, expression of the E2-regulated progesterone receptor gene was sustained in younger and in older females suggesting that age-related changes in estrogen responsiveness in the cerebral cortex are not due to the absence of ERα protein.

Keywords: estrogen receptor alpha, progesterone receptor, cerebral cortex, aging

1. Introduction

17β-estradiol (E2) is a steroid hormone that regulates gene expression in the reproductive tract as well as non-reproductive tissues including the cardiovascular, skeletal, and nervous systems (Dubey et al 2005, Nakamura et al 2007, McEwen and Alves 1999, Humphreys et al 2014). These tissues respond to E2 in part because they express the estrogen receptor (ER). ERα and ERβ are members of the steroid hormone receptor superfamily and are responsible for mediating the classical, genomic responses to E2 in target tissues (Nilsson et al 2001).

In the brain, E2 regulates sexual development and differentiation, synaptogenesis, learning and memory, mood, auditory perception, and neuroprotection (McCarthy 2008, Gillies and McArthur 2010, McEwen and Woolley 1994, Charitidi et al 2010, Fink et al 1996, Wise et al 2001, Suzuki et al 2009, Dubal et al 2001, Dubal et al 2006). A number of brain regions have been reported to respond to E2 including the hypothalamus, hippocampus, amygdala, central auditory system, and cerebral cortex (McEwen and Alves 1999, Humphreys et al 2014, Charitidi et al 2010, McEwen et al 2012, Young et al 2013).

ERα has several vital functions in the cerebral cortex. For example, it is important for neurogenesis (Suzuki et al 2007) and protects the cerebral cortex from ischemia-induced injury (Dubal et al 2001, Rau et al 2003). ERα also plays an important role in executive functioning of the prefrontal cortex, which includes working memory, attention, and behavioral inhibition (Keenan et al 2001).

A number of studies have examined ERα mRNA levels in the cerebral cortex (Wilson et al 2002, Prewitt and Wilson 2007, Westberry and Wilson 2012, Sharma and Thakur 2006). ERα mRNA is highly expressed in the cerebral cortex shortly after birth but then decreases sharply beginning by postnatal day 10 (P10) (Wilson et al 2002, Prewitt and Wilson 2007, Westberry and Wilson 2012). ERα mRNA has also been detected in the cerebral cortices of aged female mice (Thakur and Sharma 2007). However, it has become clear that mRNA levels may not accurately reflect the protein levels present (Lewandowski and Small 2005, Vogel and Marcotte 2012).

The studies that have examined the expression of ERα protein in the cerebral cortex have generally included one or two time points (Prewitt and Wilson 2007, Sharma and Thakur 2006, Kritzer 2002, Mitra et al 2003, Merchenthaler et al 2004, Naugle et al 2014). For example, Merchenthaler et al (Merchenthaler et al 2004) used autoradiographic and immunohistochemical analyses to examine the distribution of ER protein in the adult female mouse brain and demonstrated that the receptor was present at low levels in the cerebral cortex in adult mice. While this study examined the spatial distribution of ERα in the cerebral cortex in great detail, it included only one time point.

In the current studies, the temporal pattern of ERα mRNA and protein expression in the cerebral cortex was followed over 18 months in intact female mice. The mRNA and protein levels of the E2-regulated progesterone receptor (PR) gene were also examined in the cerebral cortex during the same time period. Our work demonstrates that although cortical ERα and PR mRNA levels dramatically decline with age, protein expression is sustained.

2. Materials and methods

2.1. Mice

C57BL/6 breeding pairs used for the generation of mouse pups (P5 and P17) and adult females (4 and 13 months) were obtained from Jackson Laboratory (Bar Harbor, ME). Retired female breeders were housed until 13 months and used as middle aged animals. 18 month old C57BL/6 females were purchased from the National Institute on Aging. All mice were maintained on a 12 h light/dark schedule with access to water and food ad libitum. All procedures were performed in accordance with guidelines of the University of Illinois at Urbana-Champaign Institutional Animal Care and Use Committee and Division of Animal Resources.

2.2. Estrous cycle staging

Four, 13, and 18 month old female mice were monitored for estrous cyclicity using vaginal cytology. Briefly, 5 μL of PBS was injected into the vagina of each mouse and then removed, spread on a glass slide, allowed to air dry, fixed, and nuclei were stained (3300-APD, Richard Allan Scientific, Pittsburgh, PA). Slides were imaged at 400x magnification using bright field illumination and the Leica DM 4000 B microscope. The stage of the estrous cycle was based on the presence or absence of leukocytes, cornified epithelial, and nucleated epithelial cells (Felicio et al 1984, Byers et al 2012). Four month old mice had at least two regular estrous cycles, the 13 month old mice had irregular estrous cycles, and the 18 month old mice were in persistent diestrus. The estrogen and progesterone plasma levels of C57BL/6 mice at various ages have been reported (Nelson et al 1992, Cai et al 2014). To minimize differences in hormone levels, all 4, 13, and 18 month old animals were sacrificed during diestrus.

2.3. RT-PCR

Mice were anesthetized by inhalation with isoflurane and decapitated. Total RNA was isolated from whole cortices using RNAqueous reagents (Ambion, Life Technologies, Austin, TX). Briefly, tissue was homogenized in Lysis Solution, ethanol was added, and this mixture was spun through a filter cartridge. The cartridge was washed 3x and total RNA was eluted from the cartridge with Elution Solution. Residual DNA was removed by incubating DNase I buffer and DNase I for 20 min at 37°C. DNases were inactivated by the addition of DNase Inactivation Reagent. RNA concentrations were measured and cDNA was synthesized using the iScript kit (Bio-Rad, Hercules, CA) as described by the manufacturer. Two μl of cDNA was combined with iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) and forward and reverse primers for ERα (5’-AGTGTCTGTGATCTTGTCCAG-3’ and 5’-TGTGTGCCTCAAATCCATCA-3’), GAPDH (5’-GGTGAGGCCGGTGCTGAGTATG-3’ and 5’-GACCCGTTTGGCTCCACCCTTC-3’), or PR (5’-CCGCCATACCTTAACTACCTGAG-3’ and 5’-TGCTGCCCTTCCATTGCC-3’) and real-time PCR was carried out using a Bio-Rad iQ5 multicolor Real-Time PCR Detection System. Standard curves were created using cDNA equivalents of 0.25, 2.5 and 25 ng of RNA and were run in triplicate with each primer set for each experiment.

2.4. Immunofluorescence imaging

Wild type P5, P17, 4 month, 13 month, and 18 month-old female mice were anesthetized by inhalation with isoflurane and decapitated. Whole brains were quickly harvested, bisected sagittally, and fixed in 4% paraformaldehyde for 18-24 h. Tissue was then transferred to a Leica ASP300 tissue processor for dehydration, clearing, and paraffin infiltration. Tissue was embedded in paraffin and 4 μm coronal sections were mounted on charged glass slides. Tissue sections were deparaffinized in xylene, rehydrated in two changes of 100% ethanol then two changes of 95% ethanol, and washed with PBS. Sections were then heated to 95-99 °C in 10 mM citric acid, pH 6.0, for 20 min for antigen retrieval and remained in the citric acid until the temperature reached ~30°C (~2 h). The sections were then washed 3x with PBS, incubated in blocking buffer (PBS with 0.05% Tween-20 and 5% normal donkey serum) for 10 min, and incubated in blocking buffer with an ERα-specific antibody (1:600, ab31312, Abcam Inc.) overnight at 4°C. The next day sections were washed 3x with PBS containing 0.1% Tween-20 (PBS-T), incubated with DyLight 649-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) for 30 min in the dark at room temperature, washed 3x with PBS-T, incubated with 4′,6-diamidino-2-phenylindole (DAPI) nucleic acid stain for 10 min at room temperature, washed 3x with PBS, and mounted with Pro-Long Gold antifade reagent (Life Technologies, Grand Island, NY). DAPI co-staining was included for each treatment to identify nuclei and ensure that similar numbers of cells were present. Control slides, which had not been exposed to primary antibody, were processed in parallel. As an additional negative control, the ERα-specific antibody ab31312 was incubated with a 10 molar fold excess of purified, full-length human ERα protein prior to incubation of the antibody with the brain tissue.

2.5. Immunofluorescent image collection and quantitation

All images were obtained with a 40x oil-immersion objective using a Leica DM 4000 B confocal microscope and imaging was performed using the Leica TCS SPE system and Application Suite Advanced Fluorescence software (Leica Microsystems, Inc., Bannockburn, IL). Detector gain and offset, laser power, and bandwidth of emission collection were kept constant for all treatments in each experiment and adjusted so that images had a full range of pixel intensities (0–255) and saturation was minimized.

Quantitative immunofluorescent analysis of ERα staining was performed by first selecting 4 representative fields from each animal at each age to determine the average intensity of the ERα-positive cells using Image Pro Plus image collection and analysis software. The lowest average intensity identified, 12, was then used as the mean intensity cutoff and the number of cells with a mean density/intensity of 12 or more (ERα positive) was compared to the number of DAPI-stained cells in 3-6 fields from 3-4 mice. The average number of cells per field at each age were 40 (P5), 17 (P17), 13 (4 months), 13 (13 months), and 14 (18 months).

2.6. Immunohistochemistry

Immunohistochemistry was performed as described for immunofluorescence except that after antigen retrieval endogenous peroxidases were blocked with 3% hydrogen peroxide for 20 min at room temperature. After three 5 min washes in PBS, sections were incubated in blocking buffer for 10 min and then incubated in blocking buffer with a PR-specific antibody (1:50, A0098, Dako, Carpinteria, CA) overnight at 4°C. Sections were washed with PBS-T 3x for 5 min each and then incubated with biotin-conjugated secondary antibody (1:200, Jackson ImmunoResearch Laboratories, Inc.) for 30 min at room temperature, followed by three 5 min washes with PBS-T. The ABC Peroxidase Staining kit (1:100 dilution of each Reagent A and B in PBS, 32020, Thermo Scientific, Rockford, IL) was applied to the sections for 30 min. After three washes with PBS, staining was visualized with peroxidase-sensitive Sigmafast 3,3’-Diaminobenzidine tablets (DAB, Sigma, St. Louis, MO) for 10 min. Sections were counterstained with 0.1% methyl green (Sigma, St. Louis, MO) for 5 min at 60°C, dehydrated in ethanol, cleared in xylene, and mounted with Permount (Fisher Scientific, Pittsburgh, PA). Control slices, which had not been exposed to primary antibody, were processed in parallel. Images were obtained at 40x using a Leica DM 4000 B confocal microscope with the Retiga 2000R digital camera and Image Pro Plus image collection and analysis software. Quantitative immunohistochemical analysis of PR staining was performed by using methyl green staining to count the total number of cells in 3-6 fields from 3-4 mice and the percent of cells expressing PR was determined.

2.7. Statistics

Combined data are expressed as the mean ± SEM. SAS version 9.3 (SAS Institute Inc., Cary, NC) was used for statistical analysis. ERα and PR transcript levels and the percent of ERα-positive cells were compared using one-way analysis of variance (ANOVA) and Tukey's post-hoc test. A p value of <0.05 was considered statistically significant (95% confidence interval).

3. Results

3.1. ERα transcript levels in the cerebral cortex throughout the female lifespan

Much of the previous work examining ERα expression in the cerebral cortex has analyzed ERα transcript levels in rodents either shortly after birth or in adult animals (Wilson et al 2002, Prewitt and Wilson 2007, Westberry and Wilson 2012, Thakur and Sharma 2007). To examine ERα expression across a broad range of ages, we measured ERα mRNA levels in intact females at five time points. As seen in Fig. 1, there was a significant decline in ERα transcript levels after P5 (F value = 49.22, p <0.0001), which is in agreement with previous work (Prewitt and Wilson 2007). Another significant decrease occurred at 13 months and was sustained through 18 months.

Fig. 1. ERα transcript levels decline in the cerebral cortex throughout the female lifespan.

Fig. 1

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Total RNA was isolated from the cortices of female mice at postnatal day 5 (P5), postnatal day 17 (P17), 4, 13, and 18 months of age. cDNA was synthesized and ERα mRNA levels were determined using quantitative real time PCR. The relative fold change was calculated using the delta-delta Ct method and each sample was normalized to the amount of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA present. The mean relative fold change at each age is shown ± SEM. One-way analysis of variance (ANOVA) and Tukey's post-hoc test was used to detect whether there were significant differences in ERα mRNA from female mice at P5 compared with mice at P17, 4, 13, and 18 (* p < 0.05) months of age.

3.2. ERα protein expression in the cerebral cortex throughout the female lifespan

While mRNA levels provide valuable information, protein, not mRNA, is the active biological moiety. Therefore, the relative ERα protein expression was examined in the cerebral cortices of female mice using immunofluorescence. To help limit variation, the same region of the cerebral cortex was examined in each animal (Fig. 2A, boxed regions). As shown earlier using brain slice cultures and whole mouse brains (Rao et al 2011, Dietrich et al 2013), ERα protein colocalized with NeuN and DAPI (Fig. 2B) demonstrating that the receptor was expressed in the nuclei of cortical neurons.

Fig. 2. ERα protein expression remains stable in the cerebral cortex throughout the female lifespan.

Fig. 2

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(A) The regions of the cerebral cortex examined in our studies are indicated in the red boxes. (B) The expression of ERα and the neuronal marker, NeuN, were examined in P17 female mice. Brain sections from female mice at (C) P5, (D) P17, (E) 4, (F) 13, or (G) 18 months of age were stained using immunofluorescence with the ERα-specific antibody ab31312. DAPI staining was included to identify cortical cell nuclei. Scale bars indicate 25 μm. (H) Relative ERα protein expression (± SEM) was quantitated by determining the percent of DAPI-stained cells that were ERα positive.

ERα protein was clearly observed in the cerebral cortices of female mice with the ERα-specific antibody ab31312 at P5 (Fig. 2C). While the DAPI staining suggested that the number and density of cells in the cortex had decreased by P17 (Fig. 2D), this synaptic pruning and subsequent cell death is a normal occurrence during the first two weeks of postnatal brain development (Burek and Oppenheim 1996, Low and Cheng 2006, Tremblay et al 2011). The expression of ERα protein in 4 month old female mice (Fig. 2E) was similar to the expression of ERα protein in P17 mice. Even in middle aged (13 months, Fig. 2F) and aged (18 month, Fig. 2G) mice, ERα protein expression was clearly present in the cerebral cortex.

Image analysis of cerebral cortices from female mice at P5, P17, 4 months, 13 months, and18 months indicated that 81%, 77%, 72%, 67%, and 70% of the total DAPI-stained cells, respectively, expressed ERα (Fig. 2H). While there was a slight decrease in the percent of cells expressing ERα over time, this decline was not statistically significant (F value = 2.03, p = 0.1029). These results demonstrate that ERα protein expression was sustained in the cerebral cortices of intact females, even in middle aged and aged mice.

Although our studies were largely confined to examining the expression of ERα in female mice, we found that cortical ERα expression in males and females was similar (Supplementary Fig. 1). These findings are consistent with a previous study that compared expression of ERα in the cerebral cortices of male and female rats (Kritzer 2002).

3.3. Validation of ERα protein expression in the cerebral cortex

Since ERα protein levels were very different from the ERα transcript levels, we wanted to be sure that the ERα antibody we utilized was specific. Figure 3A shows the typical pattern and intensity of ERα staining that was observed when a section of the cerebral cortex was incubated with primary and secondary antibodies. However, when the primary antibody was incubated with purified, full-length human ERα protein and then added to brain slices from the same mouse, no staining was observed (Fig. 3B) demonstrating that the primary antibody that we had used was specific for ERα. Likewise, when the primary antibody was omitted and only the secondary antibody was used, no staining was observed (Fig. 3C). Thus, the primary antibody we used was specific for ERα and did not recognize other epitopes.

Fig. 3. ERα protein expression in the cerebral cortex is validated.

Fig. 3

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Brain sections from 13 month old female mice were incubated with the ERα-specific antibody ab31312 that (A) had not or (B) had been pre-incubated with full-length (FL) human ERα protein. (C) The primary antibody was omitted so that only the secondary antibody was used. Scale bars indicate 25 μm.

3.4. Expression of an E2-regulated gene in the cerebral cortex

We previously demonstrated that ERα is a potent regulator of the E2-responsive progesterone receptor (PR) gene (Petz and Nardulli 2000, Petz et al 2002, Schultz et al 2003, Petz et al 2004b, Petz et al 2004a, Schultz et al 2005, Curtis et al 2007, Curtis et al 2009, Boney-Montoya et al 2010) and other laboratories have demonstrated that E2 and ERα are involved in increasing PR expression in the brain (Gonzales et al 2012, Moffatt et al 1998). To determine whether ERα was functional at each of the ages examined, we evaluated PR mRNA and protein expression levels in the cerebral cortices of female mice from P5 through 18 months of age. PR mRNA levels declined slightly, but not significantly, from P5 to P17 and then increased at 4 months (Fig. 4A). At 13 and 18 months, PR mRNA expression significantly declined to levels below those observed at P5 (F value = 8.95, p = 0.0008). To examine PR protein levels in female mice at each age, immunohistochemistry was performed using a PR-specific antibody and demonstrated that PR protein was present at each of the ages examined (Fig. 4B-F). Image analysis of methyl green-stained cells demonstrated that there was no statistical difference in the percent of cells that expressed PR at any of the ages examined (Fig. 4G, F value = 0.5323, p = 0.71). These findings suggest that the ERα protein residing in the cerebral cortex at each of the ages we examined was functional and helped to sustain PR protein expression.

Fig. 4. PR transcript levels decline but PR protein is present in the cerebral cortex throughout the female lifespan.

Fig. 4

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(A) Total RNA was isolated from the cortices of female mice at P5, P17, 4 mo, 13 mo, and 18 mo. cDNA was synthesized and PR mRNA levels were determined using quantitative real time PCR. The relative fold change was calculated using the delta-delta Ct method and each sample was normalized to the amount glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA present. The mean relative fold change at each age is shown ± SEM. One-way analysis of variance (ANOVA) and Tukey's post-hoc test was used to detect significant differences in PR mRNA from female mice at P5 compared with mice at P17, 4, 13, and 18 (* p < 0.05) months of age. Brain sections from (B) P5, (C) P17, (D) 4 month, (E) 13 month, and (F) 18 month old female mice were stained using immunohistochemistry with a PR-specific antibody to examine the cerebral cortex. Scale bars indicate 25 μm. (G) Relative PR protein expression (± SEM) was quantitated by determining the percent of methyl green-stained cells that were PR positive.

4. Discussion

Because ERα mediates many of the cellular responses of the cerebral cortex to E2 such as cognition (Keenan et al 2001) and neuroprotection (Dubal et al 2001, Suzuki et al 2007, Rau et al 2003), this protein plays a critical role in neurological function. While a number of studies have examined ERα mRNA levels in the cerebral cortex, there has been some question about whether ERα protein is present (Prewitt and Wilson 2007, Sharma and Thakur 2006, Kritzer 2002), especially in older females. We have now demonstrated three important findings. First, although ERα mRNA levels in the cerebral cortices of female mice decline after P5, ERα protein levels are maintained through 18 months of age. Second, like ERα, PR protein expression is sustained in the mouse cerebral cortex in spite of the decline in PR mRNA levels over time. Thus, the levels of ERα and PR protein cannot be predicted from their mRNA levels and must be measured directly. Third, the sustained expression of PR suggests that the ERα protein residing in the cerebral cortex is functional.

Expression of ERα mRNA

Although the expression of ERα has been examined in the hypothalamus (Funabashi et al 2000) and hippocampus (Adams et al 2002, Mehra et al 2005) in detail, far less is known about the expression of ERα in the cerebral cortex. Our studies focused on the cerebral cortex since this region of the brain is more susceptible to ischemic damage than other brain regions (Wise et al 2001, Suzuki et al 2009, Suzuki et al 2007, Wise et al 2000, Fukuda et al 2000) and we were interested in determining whether ERα might be present and capable of mediating estrogen responsiveness in the cerebral cortices of older animals.

Our work demonstrates that ERα mRNA levels declined with age in intact female mice, most notably after P5 and is consistent with data from previous studies (Wilson et al 2002, Prewitt and Wilson 2007, Westberry and Wilson 2012, Wilson et al 2011). However, in contrast to earlier studies in which ERα mRNA levels were very low or not detected (Wilson et al 2002, Prewitt and Wilson 2007, Westberry and Wilson 2012, Wilson et al 2011), we found that ERα mRNA transcripts were detected at each of the ages examined.

Expression of ERα protein

A number of studies have demonstrated that mRNA levels are not necessarily reflected at the level of the protein (Lewandowski and Small 2005, Vogel and Marcotte 2012). Thus, although unanticipated, it should not be surprising that the ERα and PR mRNA levels were not directly related to their protein levels. We and others have demonstrated that the half lives of unliganded ERα and PR proteins are longer than the ligand-occupied receptors (Nardulli and Katzenellenbogen 1988, Lonard et al 2000). Thus, as the hormone levels decline with age, one would anticipate that the receptor half lives would be longer and that this decreased receptor turnover could contribute to maintaining ERα and PR protein levels in older animals.

The disparity in the levels of ERα mRNA and protein that we detected could also be derived in part from the methodologies that were utilized to assess expression. While the entire cerebral cortex was utilized to monitor ERα mRNA levels, a more defined region of the cerebral cortex was used to examine ERα protein levels (Fig. 2A). A growing body of evidence from rodents and non-human primates has suggested that ERα protein expression is region-specific (Sharma and Thakur 2006, Kritzer 2002, Mitra et al 2003, Merchenthaler et al 2004, Naugle et al 2014, Behl 2002). Thus, it seems possible that the expression of ERα in the cerebral cortex as a whole may differ from the expression of this receptor in more discrete cortical regions.

In contrast to the studies reported herein, Cai et al recently reported that ERα protein levels are substantially lower in 22 month than in 3 month old female mice (Cai et al 2014). While it is possible that ERα levels may decline from 18 to 22 months of age, the hormone supplementation and resulting ligand-induced turnover may have also contributed to the reduced ERα protein they observed.

Expression of PR

We previously demonstrated that ERα is a potent stimulator of PR gene expression, that ERα binds to 8 regions of the PR gene from 311 kb upstream to 4 kb downstream of the PR-B transcription start site, and that ERα interacts with proteins bound to 4 AP1 and Sp1 sites in the proximal PR promoter (Petz and Nardulli 2000, Petz et al 2002, Schultz et al 2003, Petz et al 2004b, Petz et al 2004a, Schultz et al 2005, Curtis et al 2007, Curtis et al 2009, Boney-Montoya et al 2010). Furthermore, although a modest increase in PR expression has been reported in ERα-null mice, it is dramatically reduced compared to E2-treated wild type mice (Moffatt et al 1998, Couse et al 1995, Kurita et al 2000). Thus, although it is not the only factor involved in regulating PR gene expression, ERα is a major regulator of PR gene expression. We have now shown that the percent of cells that express PR is sustained in the cerebral cortices of female mice over 18 months suggesting that not only is ERα present, but that it is capable of helping to maintain PR protein levels. It should be noted that developmental and regional differences have been noted in the ability of E2 to increase PR protein expression (Gonzales et al 2012) as well as E2-induced spinogenesis and neuroprotection (Wise et al 2001, Suzuki et al 2009, Young et al 2013, Suzuki et al 2007, Wise et al 2000, Fukuda et al 2000).

Other contributing factors

Although our studies focused on ERα, ERβ and GPER1 are also involved in mediating the effects of estrogen in the brain. ERβ plays a role in migration of cortical neurons during development (Wang et al 2003) and although ERβ is not directly involved in reducing ischemia- mediated damage (Wise et al 2001, Dubal et al 2006), there is evidence to suggest that it plays a role in the neurogenesis that occurs after an ischemic event (Suzuki et al 2007). E2 may also elicit rapid signaling by interacting with the membrane receptor GPER1 (Raz et al 2008, Filardo et al 2002), which has been identified in several brain regions (Naugle et al 2014, Hazell et al 2009). In addition, cortical astrocytes, which express ERα, may also influence neuronal cell function (Arimoto et al 2013).

Earlier studies demonstrated that E2 protects the cerebral cortices of middle aged animals from ischemia-induced injury to the same extent as younger animals (Wise et al 2001, Dubal and Wise 2001, Wise and Dubal 2000). However, because there was some uncertainty about whether ERα protein was present in older animals, the mechanism by which E2 could mediate its effects was unclear. We have now demonstrated that ERα protein expression is maintained in the cerebral cortices of older animals. Our findings combined with previous studies (Wise et al 2001, Dubal et al 2001, Rau et al 2003, Dubal and Wise 2001, Wise and Dubal 2000) suggest that ERα may help to mediate the neuroprotective effects of E2 in the cerebral cortices of younger as well as older animals.

Supplementary Material

02

Highlights.

  • Although ERα protein levels are stable up to 18 months, ERα mRNA levels decline.

  • ERα mRNA levels cannot be used to predict ERα protein levels in the cerebral cortex.

  • PR gene expression is sustained in middle aged and older female mice.

  • PR mRNA levels cannot be used to predict PR protein levels in the cerebral cortex.

Acknowledgements

We thank Yvonne Ziegler for technical assistance and helpful discussions. This research was supported by NIH grant R01DK 053884 (to AMN). AKD was supported by a predoctoral fellowship from the NIEHS Reproductive Toxicology Training Grant T32 ES007326.

Footnotes

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Disclosure statement: The authors have nothing to disclose.

References

  1. Adams MM, Fink SE, Shah RA, Janssen WG, Hayashi S, Milner TA, McEwen BS, Morrison JH. Estrogen and aging affect the subcellular distribution of estrogen receptor-alpha in the hippocampus of female rats. J. Neurosci. 2002;22:3608–3614. doi: 10.1523/JNEUROSCI.22-09-03608.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arimoto JM, Wong A, Rozovsky I, Lin SW, Morgan TE, Finch CE. Age increase of estrogen receptor-alpha (ERalpha) in cortical astrocytes impairs neurotrophic support in male and female rats. Endocrinology. 2013;154:2101–2113. doi: 10.1210/en.2012-2046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Behl C. Oestrogen as a neuroprotective hormone. Nat. Rev. Neurosci. 2002;3:433–442. doi: 10.1038/nrn846. [DOI] [PubMed] [Google Scholar]
  4. Boney-Montoya J, Ziegler YS, Curtis CD, Montoya JA, Nardulli AM. Long-range transcriptional control of progesterone receptor gene expression. Mol. Endocrinol. 2010;24:346–358. doi: 10.1210/me.2009-0429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burek MJ, Oppenheim RW. Programmed cell death in the developing nervous system. Brain Pathol. 1996;6:427–446. doi: 10.1111/j.1750-3639.1996.tb00874.x. [DOI] [PubMed] [Google Scholar]
  6. Byers SL, Wiles MV, Dunn SL, Taft RA. Mouse estrous cycle identification tool and images. PLoS One. 2012;7:e35538. doi: 10.1371/journal.pone.0035538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cai M, Ma YL, Qin P, Li Y, Zhang LX, Nie H, Peng Z, Dong H, Dong HL, Hou WG, Xiong LZ. The loss of estrogen efficacy against cerebral ischemia in aged postmenopausal female mice. Neurosci. Lett. 2014;558:115–119. doi: 10.1016/j.neulet.2013.11.007. [DOI] [PubMed] [Google Scholar]
  8. Charitidi K, Frisina RD, Vasilyeva ON, Zhu X, Canlon B. Expression patterns of estrogen receptors in the central auditory system change in prepubertal and aged mice. Neuroscience. 2010;170:1270–1281. doi: 10.1016/j.neuroscience.2010.08.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Couse JF, Curtis SW, Washburn TF, Lindzey J, Golding TS, Lubahn DB, Smithies O, Korach KS. Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene. Mol. Endocrinol. 1995;9:1441–1454. doi: 10.1210/mend.9.11.8584021. [DOI] [PubMed] [Google Scholar]
  10. Curtis CD, Likhite VS, McLeod IX, Yates JR, Nardulli AM. Interaction of the tumor metastasis suppressor nonmetastatic protein 23 homologue H1 and estrogen receptor alpha alters estrogen-responsive gene expression. Cancer Res. 2007;67:10600–10607. doi: 10.1158/0008-5472.CAN-07-0055. [DOI] [PubMed] [Google Scholar]
  11. Curtis CD, Thorngren DL, Ziegler YS, Sarkeshik A, Yates JR, Nardulli AM. Apurinic/apyrimidinic endonuclease 1 alters estrogen receptor activity and estrogen-responsive gene expression. Mol. Endocrinol. 2009;23:1346–1359. doi: 10.1210/me.2009-0093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dietrich AK, Humphreys GI, Nardulli AM. 17beta-estradiol increases expression of the oxidative stress response and DNA repair protein apurinic endonuclease (Ape1) in the cerebral cortex of female mice following hypoxia. J. Steroid Biochem. Mol. Biol. 2013;138:410–420. doi: 10.1016/j.jsbmb.2013.07.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dubal DB, Rau SW, Shughrue PJ, Zhu H, Yu J, Cashion AB, Suzuki S, Gerhold LM, Bottner MB, Dubal SB, Merchanthaler I, Kindy MS, Wise PM. Differential modulation of estrogen receptors (ERs) in ischemic brain injury: a role for ERalpha in estradiol-mediated protection against delayed cell death. Endocrinology. 2006;147:3076–3084. doi: 10.1210/en.2005-1177. [DOI] [PubMed] [Google Scholar]
  14. Dubal DB, Wise PM. Neuroprotective effects of estradiol in middle-aged female rats. Endocrinology. 2001;142:43–48. doi: 10.1210/endo.142.1.7911. [DOI] [PubMed] [Google Scholar]
  15. Dubal DB, Zhu H, Yu J, Rau SW, Shughrue PJ, Merchenthaler I, Kindy MS, Wise PM. Estrogen receptor alpha, not beta, is a critical link in estradiol-mediated protection against brain injury. Proc. Natl. Acad. Sci. U. S. A. 2001;98:1952–1957. doi: 10.1073/pnas.041483198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dubey RK, Imthurn B, Barton M, Jackson EK. Vascular consequences of menopause and hormone therapy: importance of timing of treatment and type of estrogen. Cardiovasc. Res. 2005;66:295–306. doi: 10.1016/j.cardiores.2004.12.012. [DOI] [PubMed] [Google Scholar]
  17. Felicio LS, Nelson JF, Finch CE. Longitudinal studies of estrous cyclicity in aging C57BL/6J mice: II. Cessation of cyclicity and the duration of persistent vaginal cornification. Biol. Reprod. 1984;31:446–453. doi: 10.1095/biolreprod31.3.446. [DOI] [PubMed] [Google Scholar]
  18. Filardo EJ, Quinn JA, Frackelton AR, Jr, Bland KI. Estrogen action via the G protein-coupled receptor, GPR30: stimulation of adenylyl cyclase and cAMP-mediated attenuation of the epidermal growth factor receptor-to-MAPK signaling axis. Mol. Endocrinol. 2002;16:70–84. doi: 10.1210/mend.16.1.0758. [DOI] [PubMed] [Google Scholar]
  19. Fink G, Sumner BE, Rosie R, Grace O, Quinn JP. Estrogen control of central neurotransmission: effect on mood, mental state, and memory. Cell. Mol. Neurobiol. 1996;16:325–344. doi: 10.1007/BF02088099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fukuda K, Yao H, Ibayashi S, Nakahara T, Uchimura H, Fujishima M, Hall ED. Ovariectomy exacerbates and estrogen replacement attenuates photothrombotic focal ischemic brain injury in rats. Stroke. 2000;31:155–160. doi: 10.1161/01.str.31.1.155. [DOI] [PubMed] [Google Scholar]
  21. Funabashi T, Kleopoulos SP, Brooks PJ, Kimura F, Pfaff DW, Shinohara K, Mobbs CV. Changes in estrogenic regulation of estrogen receptor alpha mRNA and progesterone receptor mRNA in the female rat hypothalamus during aging: an in situ hybridization study. Neurosci. Res. 2000;38:85–92. doi: 10.1016/s0168-0102(00)00150-4. [DOI] [PubMed] [Google Scholar]
  22. Gillies GE, McArthur S. Estrogen actions in the brain and the basis for differential action in men and women: a case for sex-specific medicines. Pharmacol. Rev. 2010;62:155–198. doi: 10.1124/pr.109.002071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gonzales KL, Quadros-Mennella P, Tetel MJ, Wagner CK. Anatomically-specific actions of oestrogen receptor in the developing female rat brain: effects of oestradiol and selective oestrogen receptor modulators on progestin receptor expression. J. Neuroendocrinol. 2012;24:285–291. doi: 10.1111/j.1365-2826.2011.02232.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hazell GG, Yao ST, Roper JA, Prossnitz ER, O'Carroll AM, Lolait SJ. Localisation of GPR30, a novel G protein-coupled oestrogen receptor, suggests multiple functions in rodent brain and peripheral tissues. J. Endocrinol. 2009;202:223–236. doi: 10.1677/JOE-09-0066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Humphreys GI, Ziegler YS, Nardulli AM. 17beta-Estradiol Modulates Gene Expression in the Female Mouse Cerebral Cortex. PLoS One. 2014;9:e111975. doi: 10.1371/journal.pone.0111975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Keenan PA, Ezzat WH, Ginsburg K, Moore GJ. Prefrontal cortex as the site of estrogen's effect on cognition. Psychoneuroendocrinology. 2001;26:577–590. doi: 10.1016/s0306-4530(01)00013-0. [DOI] [PubMed] [Google Scholar]
  27. Kritzer MF. Regional, laminar, and cellular distribution of immunoreactivity for ER alpha and ER beta in the cerebral cortex of hormonally intact, adult male and female rats. Cereb. Cortex. 2002;12:116–128. doi: 10.1093/cercor/12.2.116. [DOI] [PubMed] [Google Scholar]
  28. Kurita T, Lee KJ, Cooke PS, Taylor JA, Lubahn DB, Cunha GR. Paracrine regulation of epithelial progesterone receptor by estradiol in the mouse female reproductive tract. Biol. Reprod. 2000;62:821–830. doi: 10.1093/biolreprod/62.4.821. [DOI] [PubMed] [Google Scholar]
  29. Lewandowski NM, Small SA. Brain microarray: finding needles in molecular haystacks. J. Neurosci. 2005;25:10341–10346. doi: 10.1523/JNEUROSCI.4006-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lonard DM, Nawaz Z, Smith CL, O'Malley BW. The 26S proteasome is required for estrogen receptor-alpha and coactivator turnover and for efficient estrogen receptor-alpha transactivation. Mol. Cell. 2000;5:939–948. doi: 10.1016/s1097-2765(00)80259-2. [DOI] [PubMed] [Google Scholar]
  31. Low LK, Cheng HJ. Axon pruning: an essential step underlying the developmental plasticity of neuronal connections. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2006;361:1531–1544. doi: 10.1098/rstb.2006.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McCarthy MM. Estradiol and the developing brain. Physiol. Rev. 2008;88:91–124. doi: 10.1152/physrev.00010.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McEwen BS, Akama KT, Spencer-Segal JL, Milner TA, Waters EM. Estrogen effects on the brain: actions beyond the hypothalamus via novel mechanisms. Behav. Neurosci. 2012;126:4–16. doi: 10.1037/a0026708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McEwen BS, Alves SE. Estrogen actions in the central nervous system. Endocr. Rev. 1999;20:279–307. doi: 10.1210/edrv.20.3.0365. [DOI] [PubMed] [Google Scholar]
  35. McEwen BS, Woolley CS. Estradiol and progesterone regulate neuronal structure and synaptic connectivity in adult as well as developing brain. Exp. Gerontol. 1994;29:431–436. doi: 10.1016/0531-5565(94)90022-1. [DOI] [PubMed] [Google Scholar]
  36. Mehra RD, Sharma K, Nyakas C, Vij U. Estrogen receptor alpha and beta immunoreactive neurons in normal adult and aged female rat hippocampus: a qualitative and quantitative study. Brain Res. 2005;1056:22–35. doi: 10.1016/j.brainres.2005.06.073. [DOI] [PubMed] [Google Scholar]
  37. Merchenthaler I, Lane MV, Numan S, Dellovade TL. Distribution of estrogen receptor alpha and beta in the mouse central nervous system: in vivo autoradiographic and immunocytochemical analyses. J. Comp. Neurol. 2004;473:270–291. doi: 10.1002/cne.20128. [DOI] [PubMed] [Google Scholar]
  38. Mitra SW, Hoskin E, Yudkovitz J, Pear L, Wilkinson HA, Hayashi S, Pfaff DW, Ogawa S, Rohrer SP, Schaeffer JM, McEwen BS, Alves SE. Immunolocalization of estrogen receptor beta in the mouse brain: comparison with estrogen receptor alpha. Endocrinology. 2003;144:2055–2067. doi: 10.1210/en.2002-221069. [DOI] [PubMed] [Google Scholar]
  39. Moffatt CA, Rissman EF, Shupnik MA, Blaustein JD. Induction of progestin receptors by estradiol in the forebrain of estrogen receptor-alpha gene-disrupted mice. J. Neurosci. 1998;18:9556–9563. doi: 10.1523/JNEUROSCI.18-22-09556.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nakamura T, Imai Y, Matsumoto T, Sato S, Takeuchi K, Igarashi K, Harada Y, Azuma Y, Krust A, Yamamoto Y, Nishina H, Takeda S, Takayanagi H, Metzger D, Kanno J, Takaoka K, Martin TJ, Chambon P, Kato S. Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts. Cell. 2007;130:811–823. doi: 10.1016/j.cell.2007.07.025. [DOI] [PubMed] [Google Scholar]
  41. Nardulli AM, Katzenellenbogen BS. Progesterone receptor regulation in T47D human breast cancer cells: analysis by density labeling of progesterone receptor synthesis and degradation and their modulation by progestin. Endocrinology. 1988;122:1532–1540. doi: 10.1210/endo-122-4-1532. [DOI] [PubMed] [Google Scholar]
  42. Naugle MM, Nguyen LT, Merceron TK, Filardo E, Janssen WG, Morrison JH, Rapp PR, Gore AC. G-protein coupled estrogen receptor, estrogen receptor alpha, and progesterone receptor immunohistochemistry in the hypothalamus of aging female rhesus macaques given long-term estradiol treatment. J. Exp. Zool. A. Ecol. Genet. Physiol. 2014;321:399–414. doi: 10.1002/jez.1871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nelson JF, Felicio LS, Osterburg HH, Finch CE. Differential contributions of ovarian and extraovarian factors to age-related reductions in plasma estradiol and progesterone during the estrous cycle of C57BL/6J mice. Endocrinology. 1992;130:805–810. doi: 10.1210/endo.130.2.1733727. [DOI] [PubMed] [Google Scholar]
  44. Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson G, Enmark E, Pettersson K, Warner M, Gustafsson JA. Mechanisms of estrogen action. Physiol. Rev. 2001;81:1535–1565. doi: 10.1152/physrev.2001.81.4.1535. [DOI] [PubMed] [Google Scholar]
  45. Petz LN, Nardulli AM. Sp1 binding sites and an estrogen response element half-site are involved in regulation of the human progesterone receptor A promoter. Mol. Endocrinol. 2000;14:972–985. doi: 10.1210/mend.14.7.0493. [DOI] [PubMed] [Google Scholar]
  46. Petz LN, Ziegler YS, Loven MA, Nardulli AM. Estrogen receptor alpha and activating protein-1 mediate estrogen responsiveness of the progesterone receptor gene in MCF-7 breast cancer cells. Endocrinology. 2002;143:4583–4591. doi: 10.1210/en.2002-220369. [DOI] [PubMed] [Google Scholar]
  47. Petz LN, Ziegler YS, Schultz JR, Kim H, Kemper JK, Nardulli AM. Differential regulation of the human progesterone receptor gene through an estrogen response element half site and Sp1 sites. J. Steroid Biochem. Mol. Biol. 2004a;88:113–122. doi: 10.1016/j.jsbmb.2003.11.008. [DOI] [PubMed] [Google Scholar]
  48. Petz LN, Ziegler YS, Schultz JR, Nardulli AM. Fos and Jun inhibit estrogen-induced transcription of the human progesterone receptor gene through an activator protein-1 site. Mol. Endocrinol. 2004b;18:521–532. doi: 10.1210/me.2003-0105. [DOI] [PubMed] [Google Scholar]
  49. Prewitt AK, Wilson ME. Changes in estrogen receptor-alpha mRNA in the mouse cortex during development. Brain Res. 2007;1134:62–69. doi: 10.1016/j.brainres.2006.11.069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rao AK, Dietrich AK, Ziegler YS, Nardulli AM. 17beta-Estradiol-mediated increase in Cu/Zn superoxide dismutase expression in the brain: a mechanism to protect neurons from ischemia. J. Steroid Biochem. Mol. Biol. 2011;127:382–389. doi: 10.1016/j.jsbmb.2011.06.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Rau SW, Dubal DB, Bottner M, Gerhold LM, Wise PM. Estradiol attenuates programmed cell death after stroke-like injury. J. Neurosci. 2003;23:11420–11426. doi: 10.1523/JNEUROSCI.23-36-11420.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Raz L, Khan MM, Mahesh VB, Vadlamudi RK, Brann DW. Rapid estrogen signaling in the brain. Neurosignals. 2008;16:140–153. doi: 10.1159/000111559. [DOI] [PubMed] [Google Scholar]
  53. Schultz JR, Petz LN, Nardulli AM. Cell- and ligand-specific regulation of promoters containing activator protein-1 and Sp1 sites by estrogen receptors alpha and beta. J. Biol. Chem. 2005;280:347–354. doi: 10.1074/jbc.M407879200. [DOI] [PubMed] [Google Scholar]
  54. Schultz JR, Petz LN, Nardulli AM. Estrogen receptor alpha and Sp1 regulate progesterone receptor gene expression. Mol. Cell. Endocrinol. 2003;201:165–175. doi: 10.1016/s0303-7207(02)00415-x. [DOI] [PubMed] [Google Scholar]
  55. Sharma PK, Thakur MK. Expression of estrogen receptor (ER) alpha and beta in mouse cerebral cortex: effect of age, sex and gonadal steroids. Neurobiol. Aging. 2006;27:880–887. doi: 10.1016/j.neurobiolaging.2005.04.003. [DOI] [PubMed] [Google Scholar]
  56. Suzuki S, Brown CM, Dela Cruz CD, Yang E, Bridwell DA, Wise PM. Timing of estrogen therapy after ovariectomy dictates the efficacy of its neuroprotective and antiinflammatory actions. Proc. Natl. Acad. Sci. U. S. A. 2007;104:6013–6018. doi: 10.1073/pnas.0610394104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Suzuki S, Brown CM, Wise PM. Neuroprotective effects of estrogens following ischemic stroke. Front. Neuroendocrinol. 2009;30:201–211. doi: 10.1016/j.yfrne.2009.04.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Suzuki S, Gerhold LM, Bottner M, Rau SW, Dela Cruz C, Yang E, Zhu H, Yu J, Cashion AB, Kindy MS, Merchenthaler I, Gage FH, Wise PM. Estradiol enhances neurogenesis following ischemic stroke through estrogen receptors alpha and beta. J. Comp. Neurol. 2007;500:1064–1075. doi: 10.1002/cne.21240. [DOI] [PubMed] [Google Scholar]
  59. Thakur MK, Sharma PK. Transcription of estrogen receptor alpha and beta in mouse cerebral cortex: effect of age, sex, 17beta-estradiol and testosterone. Neurochem. Int. 2007;50:314–321. doi: 10.1016/j.neuint.2006.08.019. [DOI] [PubMed] [Google Scholar]
  60. Tremblay ME, Stevens B, Sierra A, Wake H, Bessis A, Nimmerjahn A. The role of microglia in the healthy brain. J. Neurosci. 2011;31:16064–16069. doi: 10.1523/JNEUROSCI.4158-11.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Vogel C, Marcotte EM. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat. Rev. Genet. 2012;13:227–232. doi: 10.1038/nrg3185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wang L, Andersson S, Warner M, Gustafsson JA. Estrogen receptor (ER)beta knockout mice reveal a role for ERbeta in migration of cortical neurons in the developing brain. Proc. Natl. Acad. Sci. U. S. A. 2003;100:703–708. doi: 10.1073/pnas.242735799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Westberry JM, Wilson ME. Regulation of estrogen receptor alpha gene expression in the mouse prefrontal cortex during early postnatal development. Neurogenetics. 2012;13:159–167. doi: 10.1007/s10048-012-0323-z. [DOI] [PubMed] [Google Scholar]
  64. Wilson ME, Rosewell KL, Kashon ML, Shughrue PJ, Merchenthaler I, Wise PM. Age differentially influences estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) gene expression in specific regions of the rat brain. Mech. Ageing Dev. 2002;123:593–601. doi: 10.1016/s0047-6374(01)00406-7. [DOI] [PubMed] [Google Scholar]
  65. Wilson ME, Westberry JM, Trout AL. Estrogen receptor-alpha gene expression in the cortex: sex differences during development and in adulthood. Horm. Behav. 2011;59:353–357. doi: 10.1016/j.yhbeh.2010.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wise PM, Dubal DB. Estradiol protects against ischemic brain injury in middle-aged rats. Biol. Reprod. 2000;63:982–985. doi: 10.1095/biolreprod63.4.982. [DOI] [PubMed] [Google Scholar]
  67. Wise PM, Dubal DB, Wilson ME, Rau SW. Estradiol is a neuroprotective factor in in vivo and in vitro models of brain injury. J. Neurocytol. 2000;29:401–410. doi: 10.1023/a:1007169408561. [DOI] [PubMed] [Google Scholar]
  68. Wise PM, Dubal DB, Wilson ME, Rau SW, Bottner M, Rosewell KL. Estradiol is a protective factor in the adult and aging brain: understanding of mechanisms derived from in vivo and in vitro studies. Brain Res. Rev. 2001;37:313–319. doi: 10.1016/s0165-0173(01)00136-9. [DOI] [PubMed] [Google Scholar]
  69. Young ME, Ohm DT, Janssen WG, Gee NA, Lasley BL, Morrison JH. Continuously delivered ovarian steroids do not alter dendritic spine density or morphology in macaque dorsolateral prefrontal cortical neurons. Neuroscience. 2013;255:219–225. doi: 10.1016/j.neuroscience.2013.09.062. [DOI] [PMC free article] [PubMed] [Google Scholar]

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