Spatiotemporal Dynamics of the Expression of Estrogen Receptors In

Molecular Psychiatry (2009) 14, 223–232 & 2009 Nature Publishing Group All rights reserved 1359-4184/09 $32.00 www.nature.com/mp ORIGINAL ARTICLE Spatiotemporal dynamics of the expression of estrogen receptors in the postnatal mouse brain N Sugiyama1, S Andersson1, R Lathe2, X Fan1, P Alonso-Magdalena1, T Schwend1, I Nalvarte1, M Warner1 and J-A˚ Gustafsson1 1Department of Biosciences and Nutrition, Karolinska Institute, Novum, Huddinge, Sweden and 2Pieta Research, Edinburgh, UK

This study reports on the spatiotemporal dynamics of the expression of estrogen receptors (ERs) in the mouse central nervous system (CNS) during the early postnatal and the peripubertal period. At postnatal day 7 (P7), neurons with strong nuclear immunostaining for both ERa and ERb1 were widely distributed throughout the brain. Sucrose density gradient sedimentation followed by western blotting supported the histochemical evidence for high levels of both ERs at P7. Over the following 2 days, there was a rapid downregulation of ERs. At P9, ERa expression was visible only in the hypothalamic area. Decline in ERb1 expression was slower than that of ERa, and ERa-negative, ERb1-positive cells were observed in the dentate gyrus and walls of third ventricle. Between P14 and P35, ERs were undetectable except for the hypothalamic area. As before P7, the ovary does not produce estrogen but does produce 5a-androstane-3b,17b-diol (3bAdiol), an estrogenic metabolite of dihydrotestosterone, we examined the effects of high levels of 3bAdiol in the postnatal period. We used CYP7B1 knockout mice which cannot hydroxylate and inactivate 3bAdiol. The brains of these mice are abnormally large with reduced apoptosis. In the early postnatal period, there was 1-week delay in the timing of the reduction in ER expression in the brain. These data reveal that the time when ERs might be activated in the brain is limited to the first 8 postnatal days. In addition, the importance of aromatase has to be reconsidered as the alternative estrogen, 3bAdiol, is important in neuronal function in the postnatal brain. Molecular Psychiatry (2009) 14, 223–232; doi:10.1038/mp.2008.118; published online 4 November 2008 Keywords: 3bAdiol; central nervous system; cytochrome P-450 enzyme system; estrogen receptor-b; neuronal development; puberty

Introduction of major depressive disorder is high, but usually prodromal symptoms occur many years before diag- Although the role of estrogen in masculinization of nosis.4 As estrogen has been thought to be involved in the male rodent brain is well established,1 its role in pathophysiology of psychiatric disorders,5 we need to feminization of the female brain is still debated. It is know its role during development. Despite these thought that estrogen feminizes the female brain needs, very few attempts have been made to investi- between birth and the onset of puberty.2 The timing gate lifetime dynamics of ERs in the central nervous of this imprinting will be limited by the expression of system (CNS),6–9 and we still cannot predict how the estrogen receptors (ERs) ERa and ERb in the brain and aging or diseased brain will respond to estrogen. the availability of ER ligands, estradiol-17b (E2, a Immunohistochemistry (IHC) of ERs has been an metabolite of testosterone), and 5a-androstane-3b, essential part of estrogen studies in the CNS. After the 17b-diol (3bAdiol, a metabolite of dihydrotestoster- discovery of ERb,10 it became essential to reexamine one). For a better understanding of sex differentiation the protein map of ERs in the CNS using antibodies of the brain, the expression pattern of ERs needs to be that can selectively recognize ERa or ERb.11 There determined. have been several reports of ERa and ERb1 localiza- In human beings, just after puberty, the rate of tion in the brain,12,13 but a profile of receptor major depressive disorder increases.3 The fourth expression with age is lacking. In the previous IHC decade of life is also a time when the occurrence rate study from our group, using a highly specific antibody against ERb1 (ERb 503 IgY), we showed a high- Correspondence: Professor J-A˚ Gustafsson, Department of Bio- level expression of ERb114 and its importance during sciences and Nutrition, Karolinska Institute, Novum, Stockholm, embryogenesis.15,16 SE 141 86, Sweden. E-mail: [email protected] Very little is known about the physiological role of Received 15 August 2008; revised 30 September 2008; accepted 3 ERb ins, a splice variant of ERb, in which a novel October 2008; published online 4 November 2008 exon encoding a peptide 18 amino acids in length is Dynamics of estrogen receptors in the brain N Sugiyama et al 224 inserted into the ligand-binding domain.17,18 The Materials and methods binding affinity of ERb ins for E2 is 10- to 15-fold Animals lower than that of ERb1. The DNA-binding domain is intact and electrophoretic mobility shift assays have All experiments were approved by Stockholm’s So¨dra shown binding ability of ERb ins to estrogen respon- Fo¨rso¨ksdjursetiska Na¨mnd and conducted in accor- sive element (ERE).19–21 It has been proposed that this dance with the guidelines for the Care and Use of receptor isoform might act as a dominant negative Laboratory Animals. Mice were maintained in a light- regulator of estrogen signaling.19,22 Interestingly, the (lights on from 0600 to 1800 hours) and temperature- controlled environment with free access to laboratory affinity of ERb ins for the antiestrogen, Tamoxifen, is À/À similar to that of ERb1.23 chow (containing soy) and water. CYP7B1 mice, on The fetal brain is very well protected from circu- a C57BL/6J background, were provided by Richard lating E2 (either coming from maternal circulation Lathe (Pieta Research, UK). The colony was main- or from siblings developing in close proximity),24 tained by breeding of heterozygous pairs. Mice were because of the presence of the high-affinity estrogen- genotyped by PCR with DNA extracted from tail binding protein a-fetoprotein (AFP), which circu- biopsies. The sex of the perinatal animals (older than lates at high concentrations in the fetal blood.25 Before E18.5) was determined by inspection of the anogen- the onset of E2 biosynthesis in the brain that ital distance or by PCR (E13.5 and E18.5) of the SRY occurs at E18.5,26–28 mRNA of ERb1 is already gene. The day after mating when vaginal plug was expressed in fetal brain as early as at E10.5.29 confirmed was designated as E0.5 and the day of birth ERb1 protein is detected at E12.5, reaches a peak at was designated as P0. E18.514 and its function can be detected from E14.5.16 Although ERb1 is essential for neuronal migration Chemicals and antibodies and cortical layering, E2 itself does not appear to The 2, 4, 6, 7-tritiated E2 (100.0 Ci mmolÀ1) was be necessary as the brain of aromatase knockout purchased from Perkin-Elmer (Waltham, MA, USA). (ArKO) mice, which cannot make E2, has not been b-Galactosidase was from Sigma-Aldrich (St Louis, reported to show major neuronal abnormalities.30,31 MO, USA). Rabbit polyclonal anti-ERa (MC20) was One explanation for the activity of ERb1 in the from Santa Cruz Biotechnology (Santa Cruz, CA, absence of E2 is the presence of an alternative ligand USA). Chicken polyclonal anti-ERb1 antibody, chicken for this receptor. polyclonal anti-ERb ins antibody, and rabbit poly- We hypothesized that estrogenic activity in the fetal clonal anti-ERbLBD antibody were produced brain is due to 3bAdiol. 3bAdiol is secreted by the in our laboratory.11 Mouse monoclonal anti-NeuN immature ovaries32–34 and testes.35 As 3bAdiol is antibody was from Chemicon (Temecula, CA, USA). synthesized in situ in cells expressing 5a-reductase Biotinylated anti-rabbit, anti-chicken, and anti-mouse and 17b-hydroxysteroid dehydrogenase type 7,36–38 its antibodies raised in goats were from Zymed (South level in the brain is not affected by AFP. In order to San Francisco, CA, USA). understand the role of 3bAdiol in the developing brain, we have examined the brains of CYP7B1 Phenotype analysis knockout mice (CYP7B1À/À). CYP7B1 is the enzyme The brain areas and the lateral ventricular size of WT responsible for hydroxylation and inactivation of the and CYP7B1À/À were measured on Nissl-stained estrogenicity of 3bAdiol. CYP7B1À/À mice cannot sections and compared at P0 and P14. Postnatal WT hydroxylate 3bAdiol and there are therefore high and CYP7B1À/À mice were weighed at P9 and P14. levels of this estrogenic steroid in the brain of these After decapitation, the brain was dissected out and its mice. wet weight was immediately measured. The number We here report an enlarged brain and decreased of animals in each group was 10–16. The mean values number of apoptotic cells in the brain of the for each genotype of the same age and sex were CYP7B1À/À mice during late fetal life. The brain size compared by Student’s t-test and given as mean± normalizes after puberty. We have investigated ERa s.e.m. and ERb1 expression profiles in the early postnatal and peripubertal period of wild-type (WT) and CYP7B1À/À mouse brains. The time when ERs may Preparation of neural tissue sections cause feminization of the brain is limited to the first 8 To obtain fetal brains, pregnant mice were deeply

postnatal days. In the frontal cortex, there is a anesthetized with CO2 and perfused with phosphate- window in time when most of the brain is insensi- buffered saline (PBS) followed by 4% paraformalde- tive to estrogen. This timing is delayed in hyde. Embryos were taken out and put on ice, and CYP7B1À/À compared with WT mice suggesting that brains were dissected and postfixed in the same 3bAdiol is involved in this mechanism and that fixative overnight at 4 1C. Offspring of mice were normal metabolism of this estrogenic steroid is decapitated on each postnatal day. Following decap- essential for control of ER expression. Our IHC data itation, the brain was removed quickly and fixed clearly show that ER expression in the brain is under using ice-cold 4% paraformaldehyde followed by 72 h strict control in a spatiotemporal manner during postfixation in the same fixative at 4 1C. The brains development. were processed for 4-mm paraffin sections.

Molecular Psychiatry Dynamics of estrogen receptors in the brain N Sugiyama et al 225 IHC of ERs gradients and centrifuged at 4 1C for 16 h at 300 000 g. IHC was performed as described before.14 In brief, Successive 100-ml fractions were collected from brain sections were deparaffinized in xylene and the bottom, and assayed by liquid scintillation rehydrated through graded ethanol. Antigens were counting. retrieved by boiling in 10 mM citrate buffer (pH 6.0). Endogenous peroxidase was quenched with 0.3% Western blotting hydrogen peroxide in 50% methanol and nonspecific Following sucrose density gradient sedimentation, binding blocked with 1% bovine serum albumin fractions were collected and analyzed by western (BSA). For ERb1 and ERb ins staining, slides were blotting for ERb1. Samples were precipitated with incubated with 0.15 units mlÀ1 of b-galactosidase for trichloroacetic acid, and the precipitate was washed 2 h at room temperature. Thereafter, sections were with methanol. Pellets were dissolved in SDS sample incubated with either one of the primary antibodies at buffer, and the proteins were resolved by SDS– appropriate dilutions in PBS containing 0.1% NP40, polyacrylamide gel electrophoresis with 4–20% gra- 1% BSA overnight. After thorough washing, sections dient gels (Invitrogen, Carlsbad, CA, USA) as were incubated for 1 h with biotinylated secondary described before.42,43 After transfer to PVDF mem- antibodies at a 1:200 dilution in PBS. The sections branes (Applied Biosystems, Foster City, CA, USA), were rinsed and incubated with avidin-biotinylated blots were probed with anti-ERbLBD antibody, fol- horseradish peroxidase complex (Vectastain Elite lowed by appropriate secondary antibodies conju- ABC kit; Vector Laboratories, Burlingame, CA, USA) gated with horseradish peroxidase. Detection was by for 1 h. The peroxidase–substrate reaction was enhanced chemiluminescence ECL kit (Amersham visualized by 3,3-diaminobenzidine (Dako, Carpen- Pharmacia, Pittsburgh, PA, USA). teria, CA, USA). The sections were slightly counter- stained with hematoxylin. In every run of staining, Quantitative real-time PCR NeuN immunostaining was included as a positive Offspring of mice were decapitated on each postnatal control for the quality checking of IHC, such as day. The brain was removed and immediately frozen fixation status of the sections. In this report on ER using dry ice powder. Only male mouse brains were IHC, we focused on male mice of both of WT and analyzed. Total RNA extraction from the whole brain CYP7B1À/À mice. samples was performed by RNseay lipid tissue midi kit (Qiagen GmbH, Germany). Genomic DNA contam- TUNEL labeling ination was removed by the treatment with DNase I The terminal deoxynucleotidyl transferase-mediated (Qiagen GmbH). First-strand cDNA synthesis from dUTP nick-end labeling (TUNEL) assay was per- total RNA was performed by SuperScript III Reverse formed as previously described.39 The brain sections transcriptase (Invitrogen). The real-time PCR was were deparaffinized, rehydrated, and immersed in performed using ABI Prism 7000 Sequence Detection proteinase K solution (20 mgmlÀ1 in 10 mM Tris-HCl, System (Applied Biosystems) on individual cDNA pH 7.4) for 30 min at 37 1C. After rinsing, sections samples from each mouse and each sample was run were incubated with TUNEL reaction mixture for 1 h in duplicate. The primers used for each gene at 37 1C(In situ cell death kit; Roche Diagnostic, were: ERa 50-GCTGCGCAAGTGTTACGAAG, 30-TCTT Mannheim, Germany). TUNEL-positive cells in the TCCGTATGCCGCCTT; ERb150-GCCAACCTCCTGA ventricular/subventricular zone were counted and the TGCTTCTT, 30-TGCCCTTGTTACTGATGTGCC; ERb mean value for each genotype, of the same age was ins 50-CGTTCTGGACAGGTCCTCAGA, 30-CCCTTGG compared at E13.5 and E15.5 by Student’s t-test, and GACAGCACTCTTC; 18S rRNA 50-GTCTGTGATGCCC given as mean±s.e.m. Four to six animals for each TTAGATG, 30-AGCTTATGACCCGCACTTAC. Detected group were analyzed. data were normalized to 18S rRNA and expressed relative to P4. The number of animals in each Sucrose density gradient sedimentation postnatal day was three. To make comparisons Sedimentation studies were carried out as described between groups, statistical analysis was performed previously.40,41 Offspring of the pregnant mice were via one-way analysis of variance (ANOVA) followed decapitated on P7. The brain was removed and frozen by Tukey–Kramer’s honestly significant difference using dry ice powder, and stored at À80 1C until the post hoc test. time of assay. Frozen brains were pulverized and added to a buffer (for MCF-7 cells the suspension was Results 100 mgmlÀ1 buffer, and for mouse brain, it was 1 g mlÀ1 buffer). Cytosol was obtained by centrifugation of the Quantification of transcript levels of ERs in WT mouse homogenate for 1 h at 4 1C at 100 000 g. Whole brain brain during development cytosol was incubated for 2 h at 4 1C with 10 nM Quantitative real-time PCR with RNA extracted from tritiated E2 in the presence or absence of excessive whole mouse brains showed that ERa,ERb1, and ERb cold E2. In some experiments, after this step, cytosol ins mRNA were high on P4. Over the next 10 days, was incubated with 1 ml MC20 antibody for 10 min on ERb transcripts declined steadily to reach a low level ice. Sucrose density gradients (10–30% (w/v) sucrose) of 50% for ERb1 and 25% for ERb ins of their levels at were prepared. Samples of 200 ml were layered on P4. ERb1 mRNA increased again by P35 in the brain,

Molecular Psychiatry Dynamics of estrogen receptors in the brain N Sugiyama et al 226 IHC of ERs in CNS of WT mouse during early postnatal and peripubertal periods At the age of P7, very strong and sharp signals of ERa, and strong to moderate, but distinct nuclear staining of ERb1 were widely distributed in the brain (Figure 2A). These IHC signals of ERs were shown to be specific, as all signals were ablated on preincuba- tion of the primary antibodies with the antigens against which they were raised. Sucrose density gradient sedimentation (Figure 2B) followed by western blotting (Figure 2C) using whole brain extracts at P7 also revealed abundant presence of both ER proteins. Two days later, at P9, ERa expression was much reduced, with only a few areas such as the hypothalamus and the amygdala remaining positive (Figures 3a–h). Diminution of ERb1 expression was slower than that of ERa. In several brain regions including the dentate gyrus of the hippocampus, the medial habenular nucleus, the thalamus and the wall of third ventricle, ERb1-positive cells were observed but ERa expression was not detected in these areas at this age (Figures 3i–p). At P14 and P35, both ERs were difficult to detect with the exception of very limited parts of the hypothalamic area such as the medial preoptic nucleus and the arcuate nucleus. Temporal changes in expression of ERs were most representative in the frontal cortex. In this brain region, there was a rapid downregulation of ERs between P7 and P9. As judged from the undetectable levels of ERs, the peripubertal period from P14 to P35 appears to be a window in time when the brain is insensitive to estrogen. Four months after birth, there was a gradual increase in the number of ERa and ERb1 positive cells (Figure 4). ERb ins protein was undetectable after birth.

ER expression in the CYP7B1À/À mouse brain Focusing on the frontal cortex, we also examined ERa and ERb1 expressions in the CYP7B1À/À mouse brain at the ages of P9, P14, and P35 (Figure 5). At P9, there were several ERa-positive cells and abundant ERb1- positive cells. After P14, ERa expression in the CYP7B1À/À mouse brain decreased, and ERb1 expression was confined to layer II of the cortex. At P 35, cells positive for ERa or ERb1 were difficult to find. However, there were still a few positive cells. This Figure 1 Developmental changes of mRNA expression of pattern is different from our observations of WT ERs. Quantitative real-time PCR was performed on total mouse brain where essentially no ERs are detected RNA isolated from the whole brain of P4, P7, P9, P14, P21, and P35 male mice to determine the mRNA expression of between P14 and P35. On the basis of immunohisto- ERa,ERb1, and ERb ins. Data were normalized to the chemical analysis, the decrease in expression of ERs À/À internal control, 18S rRNA, and expressed relative to P4. seemed to be delayed in CYP7B1 mice; ER Value represents the mean and s.e.m. Asterisk (*) indicates expression in CYP7B1À/À mouse brains at P9, P14, the significant differences from P4 (P < 0.05 by one-way and P35 was quite similar to that in WT mice at P7, analysis of variance (ANOVA) followed by the Tukey– P9, and P14, respectively (Figures 4 and 5). Kramer’s honestly significant difference (HSD) post-hoc test); n = 3 for each group. Transient enlargement of forebrain in postnatal CYP7B1À/À mouse Nissl staining of coronal brain sections revealed an but ERb ins remained low. Postnatal decline in ERa increase in the size of the forebrain area and mRNA was more modest with only a 20% decrease in compression of the lateral ventricles in the the first two weeks of postnatal life (Figure 1). CYP7B1À/À mice between P0 and P14 in both sexes

Molecular Psychiatry Dynamics of estrogen receptors in the brain N Sugiyama et al 227

Figure 2 Expression of estrogen receptor a (ERa) and estrogen receptor b1 (ERb1) in the mouse brain at P7. (A) Representative Immunohistochemistry (IHC) of ERa (a, c, and e) and ERb1(b, d, and f). The right panels are adjacent sections of the left panels from the same brains. Cell nuclei that are negative for ERs are made evident by the blue counterstain. (B) Sucrose density gradient sedimentation profiles of ERs from the cytosol of male mouse brain at P7 (’) and of the same cytosol pre-incubated with MC20 (anti-ERa antibody) (m). Samples were pre-incubated with tritiated E2 (10 nM) and radioactivity was measured by scintillation counting. The sucrose gradient shows a peak at fraction 23. Pre-incubation with MC20 (anti-ERa antibody) reduces the peak at fraction 23 (m) indicating that this fraction contains both ERa and ERb1. (C) Western blot detection of ERb1 using the fraction pre-incubated with MC20 (m). Presence of ERb1 in fraction 23 was confirmed by western blotting showing a band at expected molecular weight (MW) of 63 kDa. The band corresponding to MW 55 kDa in fraction 13 and surrounding fractions corresponds to reaction of the secondary antibody with the added anti ERa immunoglobulin G (IgG). LSD: lateral septal nucleus, dorsal part; Pir: piriform cortex; VDB: nucleus of the vertical limb of the diagonal band. Size bar = 50 mm.

(Figure 6). The wet weights of the brains of CYP7B1À/À can be used to identify receptor-expressing cells mice were higher than those of WT for both sexes at without losing in situ information. This is especially P9 and P14, although there was no difference in body important in the CNS where positioning of neurons is weight between CYP7B1À/À and WT mice (Figure 7). closely connected to function. As receptor functions By 3 months of age, the size and shape of the brains of are mediated by the receptor protein not mRNA, the CYP7B1À/À mice had normalized, and were indis- protein needs to be measured. Localization of mRNA tinguishable from WT mice (Figure 6). At E13.5 and is useful but not sufficient because the level of mRNA E15.5, there were fewer apoptotic cells in the does not always reflect that of receptor protein. Our ventricular/subventricular zone of CYP7B1À/À mice mRNA data determined by real-time PCR show that than in WT mice (Figure 8). with ERb1 and ERb ins the mRNA roughly parallels the protein but this is not the case with ERa. Changes in ERa mRNA expression between birth and puberty Discussion were small, although there are dynamic changes of Reliable IHC staining for ERs in the CNS has been ERa IHC in the frontal cortex during the same period. very important for understanding the role of estrogen The role of ERb ins in the brain during the in sex differentiation, in regulating the neuroendo- peripubertal period is also of great interest. Handa crine hypothalamus and pituitary axis, and in clinical et al.22 have hypothesized that ERb ins may act as a studies on effects of hormone replacement therapy dominant negative, low-affinity ER in the brain. Their (HRT) for menopausal syndrome, cerebral ischemic hypothesis is based on their IHC observation of attack, and neurodegenerative disorders. Although colocalization of ERb1 and ERb ins in the preoptic IHC is regarded as descriptive, it is the only tool that area, the bed nucleus of stria terminalis, and the

Molecular Psychiatry Dynamics of estrogen receptors in the brain N Sugiyama et al 228 amygdala of the adult rat brain. These are brain areas variant of ERb may be playing some modulatory role that are highly sensitive to circulating E2. If Handa during embryonic life. One such function could be et al.22 are correct, our result that ERb ins mRNA protection of the fetus from circulating high-level E2 decreases just after birth, suggests that this splice by being a dominant repressor of both ERa and ERb1.

Figure 4 Strongly and rapidly downregulated expression of estrogen receptor a (ERa) (left panels) and estrogen receptor b1 (ERb1) (right panels) in the developing mouse forebrain. Expression of ERs in the frontal cerebral cortex (area ranging from the cingulate cortex to the secondary motor cortex) was examined throughout development. Significant reduction of ERa was observed between P7 and P9. From P9, ERa-ir cells were undetectable in these regions. Widely distributed ERb1-ir cells at P7 were confined to the layer II by P9. Between P9 and P14, ERb1- ir was lost. After 4 months of age, immunoreactivities of ERa and ERb1 reappear. Size bar = 50 mm.

Figure 3 Much reduced expression of estrogen receptor a (ERa)(a–o) and estrogen receptor b1 (ERb1) (b–p) in the mouse brain at P9. Both ERa-ir and ERb1-ir cells were observed in a limited part of cortex (a and b), hypothalamus (c–f), and the amygdala (g and h). In several brain regions, ERb1 selective expression was observed (i–p). The right panels are adjacent sections of the left panels from the same brains. 3V: 3rd ventricle; Arc: arcuate nucleus; DG: the dentate gyrus of the hippocampus; MePD: medial amygda- loid nucleus, posterodorsal part; MHb: medial habenular nucleus; VMN: ventromedial nucleus of the hypothalamus; S2: secondary somatosensory cortex. Size bar = 50 mm.

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Figure 5 Delayed timing of downregulation of estrogen receptor a (ERa) (left panels) and estrogen receptor b1 (ERb1) (right panels) in CYP7B1À/À mice. Strong signals of ERa-ir and ERb1-ir cells were observed in all layers of cerebral cortex at P9. At P14, ERa-ir and ERb1-ir cells were confined to the layer II. At P35, numbers of ERa and ERb1 positive cells were much reduced; however, a few scattered positive cells were still observed. Size bar = 50 mm.

Figure 6 Increased postnatal brain size in CYP7B1À/À This function would complement the function of mice. Nissl staining of forebrain sections showed larger AFP. size of areas and compressed lateral ventricles of CYP7B1À/À ERb1 expression has been reported in several brain (right) compared with wild-type (left) mice at P0 (upper regions such as the hippocampus and the raphe panels) and P14 (middle panels). Note the compression of nucleus in the midbrain44 which were previously the lateral ventricles with little empty space left in CYP7B1À/À mice at P0 and P14. At 3 months of age, brain thought to be estrogen nonresponsive. In the present size of CYP7B1À/À was normalized and showed no differ- study, we report on the dynamic spatiotemporal ence compared with wild type (lower panels). Size changes of the two ERs in the late period of CNS bar = 0.5 mm. development. The high level of ER expression found in the fetal brains is maintained until P7. Thereafter, receptor levels diminish rapidly and by P9, ERa It has previously been shown that during postnatal expression is confined to the hypothalamic regions. development in male rodent brain, there is a rapid ERb1 declines more slowly between P7 and P14. At rise in cell death, with a peak on P7 followed by a P9, ERb1 was still expressed in the cortex, but mainly sharp decline to negligible levels by P15. In females, in the layer II. there is a less sharp increase in cell death but the We also show that there is a window in time period of increased apoptosis is longer and lasts until between P14 and P35 when there is neither ERa nor P25.45 The apoptosis coincides with the loss of ERb1 in most of the brain. Thus, in the peripubertal expression of interleukin (IL)-9, a protective cytokine period most of the brain appears to be unresponsive to in the CNS. IL-9 and IL-9 receptor mRNA levels in the estrogen. During development and peripubertal per- brain peak on early postnatal days followed by a iod of life, it may be that some organs require E2 dynamic decline by P10.46 The similar temporal whereas other organs do not need E2, or need to avoid decline in ERs and IL-9 suggests a common regu- E2. This may be particularly true in an elaborate lation. composite organ such as the brain, in which different In the developing CNS, estrogen has been shown to types of neurons exist close to each other, and where have at least two important roles; sex differentiation E2 may elicit completely opposite effects on different and cortical neuronal migration. Despite major ad- neurons. It is quite natural to assume that estrogen vances in understanding neuronal development and signaling is modified not only by the level of E2 itself neuroendocrinology, the hormonal regulation of the but also by the differential expression levels of ERa developing brain is still insufficiently understood. and ERb so that each cell can translate one serum The discovery of ERb and establishment of IHC for concentration of E2 in different suitable ways. ERb1 and ERb ins have brought us new tools to

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Figure 7 Increased brain wet weight of postnatal CYP7B1À/À mice. Tissue wet weights are expressed as the mean and s.e.m. The brain wet weight of CYP7B1À/À mice was significantly increased for both sexes at P9 and P14 as compared with wild type (WT). No differences in body weight of the CYP7B1À/À as Figure 8 Decreased cell death in CYP7B1À/À mice. (a)At compared with WT could be found for either males or females E15.5, fewer TUNEL-positive cells were seen in the at both ages. *P < 0.05 versus WT of the same sex and same ventricular/subventricular zone of CYP7B1À/À mice than age; n = 10–16 for each groups. in wild-type (WT) mice. The border of the lateral ventricles is outlined in gray. Size bar = 50 mm. (b) The number of apoptotic cells was compared by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) increase our insight into this important aspect of assay at E13.5 and E15.5. TUNEL-positive cells in the brain biology. ventricular/subventricular zone are expressed as the mean and s.e.m. *P < 0.05 versus WT of the same age; n = 4–6 for Our observation of rapid changes in ER expression each groups. provokes another important reflection: 2 days in the life of a mouse is the equivalent of 1–2 months of a human being. Although at birth, the state of matura- tion of the brain is much different in mouse and man, that were caused by an excessive amount of 3bAdiol it is reasonable to assume that during postnatal prepubertally. This is consistent with a previous development changes in ERs also occur in the human finding that CYP7B1 is active in the brain only during brain. Changes in hormone levels might well influ- early development.49 ence brain ER expression and if it does, it can be Our data indicate that excessive amounts of anticipated that there would be changes in ER 3bAdiol inhibit the normal control of expression of expression in the brains not only at birth and puberty ERs suggesting that 3bAdiol has an important role in but also during the menopausal period and in aging. the regulation of ER expression during development. In rodents, the neuroprotective effect of E2 in the To our knowledge, animal models that show abnor- brain has been shown to be different depending on mally large, heavy brain are rare. Krieglstein et al.50 the timing of administration of E2 after ovariectomy.47 reported that immunoneutralization of endogenous In this regard, the data generated in the recent HRT transforming growth factors-b (TGF-b) prevented trials48 have to be reevaluated to include considera- ontogenetic neuronal death in the chicken embryo. tion of the age of subjects selected for HRT. The One possible scenario in the developing brain is that response of a 70- to 80-year-old woman who has not constant activation of ERb1 stimulates production of previously had HRT cannot be expected to be similar inhibin and this inhibin blocks the TGF-b signaling. to that of a woman at menopause. Thus, the CYP7B1À/À mouse resembles the mouse in The brain phenotype in CYP7B1À/À mice strongly which TGF-b is neutralized. One other possible suggests that ER signaling is involved in the physio- reason for enlargement of CYP7B1À/À brain could be logical regulation of developmental apoptosis and that ERs might be regulators of IL-9 signaling and determination of fetal brain size. Interestingly, the 1-week delay of downregulation of ERs may cause the bigger brain size in CYP7B1À/À mice is normalized same delay of IL-9 downregulation. after puberty. It appears that after puberty, compensa- The astonishing fact that the ArKO mouse does not tory systems correct the disturbances in ER regulation show any severe neuronal abnormality reinforces the

Molecular Psychiatry Dynamics of estrogen receptors in the brain N Sugiyama et al 231 suggestion that knockout of aromatase does not mean depression. The Baltimore epidemiologic catchment area follow- complete absence of estrogen. We propose that up. Arch Gen Psychiatry 1997; 54: 993–999. 3bAdiol might be a main estrogen in the brain 5 Seeman MV. Psychopathology in women and men: focus on female especially during development. As early as 1974, hormones. Am J Psychiatry 1997; 154: 1641–1647. 33 6 Zsarnovszky A, Belcher SM. Identification of a developmental 3bAdiol was reported to be estrogenic. In our recent gradient of estrogen receptor expression and cellular localization studies, we have shown that 3bAdiol is a strong in the developing and adult female rat primary somatosensory binder of ERb141 and that CYP7B1 is the enzyme that cortex. Brain Res Dev Brain Res 2001; 129: 39–46. regulates ERb1 function by regulating the level of 7 Perez SE, Chen EY, Mufson EJ. 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