Involvement of Estrogen Receptor &Beta

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ORIGINAL ARTICLE Involvement of estrogen receptor b in maintenance of serotonergic neurons of the dorsal raphe

H Suzuki1, RPA Barros1, N Sugiyama1, V Krishnan2, BC Yaden3, H-J Kim1, M Warner1,4 and J-Å Gustafsson1,2

The serotonergic neurons of the dorsal raphe (DR) nucleus in the CNS are involved in fear, anxiety and depression. Depression and anxiety occur quite frequently in postmenopausal women, but estrogen replacement to correct these CNS disorders is at present not favored because estrogen carries with it an increased risk for breast cancer. Serotonin synthesis, release and reuptake in the DR are targets of pharmaceuticals in the treatment of depression. In the present study we have examined by immunohistochemistry, the expression of two nuclear receptors, that is, the estrogen receptors ERa and ERb. We found that ERb but not ERa is strongly expressed in the DR and there is no sex difference and no change with ageing in the number of tryptophan hydroxylase (TPH)-positive neurons in the DR of wild-type (WT) mice. However, in ovariectomized (OVX) WT and in ERbÀ/À mice, there was a marked reduction in the number of TPH-positive normal-looking neurons and a marked increase in TPH-positive spindle-shaped cells. These neuronal changes were prevented in mice 1--3 weeks (but not 10 weeks) after OVX by the selective ERb agonist, LY3201, given as continuous release pellets for 3 days. The ERb agonist had no effects on glucose homeostasis. Thus, the onset of action of the ERb agonist is rapid but there is a limited window in time after estrogen loss when the drug is useful. We conclude that, rather than estradiol, ERb agonists could be useful pharmaceuticals in maintaining functional DR neurons to treat postmenopausal depression.

Molecular Psychiatry (2013) 18, 674--680; doi:10.1038/mp.2012.62; published online 5 June 2012 Keywords: LY3201; postmenopausal depression; tryptophan hydroxylase

INTRODUCTION ligand, 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), is diabeto- 13 Estrogen receptor (ER) b is widely expressed in the developing genic, we have examined the effects of LY3201 on glucose mouse brain and is essential for the correct layering of the homeostasis. cortex.1--3 With immunohistochemical techniques, ERb expression has been studied in several laboratories.4 There is little or no ERb expression in adult pituitary5,6 or in the endometrium,5 whereas in MATERIALS AND METHODS the mammary gland ERb is abundant and has antiproliferative, Animals pro-differentiative effects.7 In contrast to developing mouse brain WT, ERbÀ/À and ERaÀ/Àmice were generated as previously described.14--16 where ERb is abundant, in adults, expression of ERb is confined to All mice were backcrossed to a C57BL6 background for at least 10 few areas of the brain. Owing to lack of ERb expression in the generations. Animals were housed on a 12-h light/dark cycle under pituitary, selective ERb agonists do not interrupt the hypothala- controlled temperature (20--22 1C) and fed a standard pellet diet with mo--pituitary--gonadal axis, nor do they cause growth of the water provided ad libitum. For brain collection, mice were deeply mammary gland or uterus.5,8 anesthetized with carbon dioxide and perfused manually using gravity It has been known for some time that ERa is not expressed in through left ventricle with phosphate-buffered saline followed by 4% the serotonergic neurons of the dorsal raphe (DR) but that paraformaldehyde. Brains were dissected and postfixed in the same estrogen influences the serotonergic system.9 For many years, the fixative overnight at 4 1C and were processed for 5-mm paraffin sections. effects of estrogen on the DR were thought to be indirect. This To examine glucose homeostasis, skeletal muscle and white adipose view had to be revised after the discovery of ERb in 1996 and the tissue were excised, frozen immediately for real-time quantitative PCR and finding in several laboratories of the expression of ERb in neurons blood was collected from the orbital sinus for serum metabolic panel 10,11 of the DR. E2 is equally potent on ERa and ERb, but agonists measurements. selective for ERa or ERb have been synthesized. We have obtained one ERb-selective agonist from the pharmaceutical company Eli Ovariectomy and treatment with E2 or LY3201 Lilly (LY3201) and shown that it is anxiolytic.12 At the age of 6 months, 45 WT mice were ovariectomized (OVX) bilaterally In the present study we have examined the expression of ERb in through a single dorsal midline incision across the lumbar region, making the DR of (wild-type) WT mice and the consequences to the DR both ovaries accessible. The ovary-attached fat pad was gently grasped to neurons of loss of ERb. In addition, we have investigated the lift and exteriorize the ovary. Subsequently, the periovarian sac was peeled effects of LY3201 on tryptophan hydroxylase (TPH) in the DR back over the surface of the ovary, allowing removal of the whole ovary. and, as we have previously reported that one ERb-seletive This surgery was performed at Jackson Laboratory (Sacramento, CA, USA).

1Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX, USA; 2Musculoskeletal Research, Lilly Research Laboratories, Indianapolis, IN, USA; 3Department of Biology, IUPUI, Indianapolis, IN, USA and 4Department of Biosciences and Nutrition, Karolinska Institutet, Novum, Stockholm, Sweden. Correspondence: Professor J-Å Gustafsson, Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, 3605 Cullen Boulevard, Houston 77204, TX, USA. E-mail: [email protected] Received 31 January 2012; revised 5 March 2012; accepted 8 March 2012; published online 5 June 2012 Estrogen receptor b in the dorsal raphe H Suzuki et al 675 1, 3 and 10 weeks after OVX, mice were randomly placed in three groups release), Innovative Research of America, Sarasota, FL, USA); and (3) five mice as follows: (1) five mice were inserted with placebo pellets; (2) five mice were were inserted with LY3201 pellets (200 mgkgÀ1 per day, Eli Lilly, Indianapolis, À1 inserted with E2 pellets (10 mgkg per day, 6.3 mg per pellet for 21-day IN, USA) subcutaneously. Hormonal treatment lasted for 3 days.

a ER b ER

Figure 1. Expression of estrogen receptors (ERs) in the dorsal raphe (DR) nucleus of 6-month-old male mice. Several ERa-positive cells are found in the periaqueductal gray (PAG) but not in the DR (a). Many ERb-positive neurons are found in the DR (b). The dashed line shows the border of the DR. The boxes represent higher magnification of immunoreactive ERa in the PAG, and of ERb in the lateral part of the DR. Scale bars are 100 mm in lower magnification and 20 mm in higher magnification.

Figure 2. Comparison of the expression of tryptophan hydroxylase (TPH) in the DR of male/female (a, b/c, d), young/aged (a, b/c, d) and (wild- type) WT/gene-deficient (a--d/e) mice (n ¼ 5). There is neither sex nor age difference of the number of TPH-positive neurons. However, there is a marked decrease of TPH in estrogen receptor (ER)bÀ/À aged male mice. Meanwhile, there is a marked increase in TPH-positive spindle- shaped cells at the lateral part of the DR of ERbÀ/À aged male mice. **Po0.01 versus WT male, 15 months (M) of age. Scale bar is 100 mmin lower magnification and 20 mm in higher magnification.

& 2013 Macmillan Publishers Limited Molecular Psychiatry (2013), 674 -- 680 Estrogen receptor b in the dorsal raphe H Suzuki et al 676 Immunohistochemistry and cell count a 1:200 dilution and incubated with avidin-biotinylated horseradish Parafﬁn-embedded sections were de-waxed in xylene followed by peroxidase complex (Vector laboratories, Burlingame, CA, USA) for 1 h for rehydration in graded concentrations of ethanol. Antigen retrieval was ERb staining. The slides were developed using DAB (Dako, Carpinteria, CA, achieved by placing slides in 97 1C citrate buffer (pH 6.0) for 10 min. USA) and counterstained with hematoxylin. To count the number of TPH-

Endogenous peroxidase was quenched by 30 min incubation in 1% H2O2 in positive cells, six serial sections that included the DR were chosen from 50% methanol followed by blocking of unspeciﬁc protein binding with each mouse, and TPH cells were counted in the entire DR and lateral part 3% bovine serum albumin for 10 min. Sections were incubated with of the DR by two independent investigators. The location of the DR and rabbit anti-ERa (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), lateral part of the DR was determined as previously described.17 chicken anti-ERb (1:200 home made) or mouse anti-TPH (1:800, Sigma- Aldrich, St. Louis, MO, USA) in 3% bovine serum albumin with phosphate- buffered saline and 0.1% NP40 at room temperature overnight. The effects of LY3201 on glucose homeostasis Corresponding HRP polymer solution (Bio care medical, Concord, CA, To observe the effects of LY3201 on glucose metabolism, WT male mice USA) was added for 30 min at room temperature for ERa and TPH staining. were administered LY3201 by oral gavage (0.35 mg kgÀ1 per day) for Sections were incubated for 1 h with biotinylated secondary antibodies at 3 days. There were 27 mice in the vehicle-treated and 27 in the LY3201-

Figure 3. At the age of 6 months, wild-type (WT) female mice were ovariectomized (OVX). At 1, 3, and 10 weeks (W) after OVX, mice were implanted subcutaneously with placebo, E2 or LY3201 pellets. The duration of hormone exposure was 3 days. There were five mice in each group. Photomicrographs represent the change in tryptophan hydroxylase (TPH) positivity in the dorsal raphe (DR) and the chart represents the TPH-positive cell numbers in whole or lateral part of the DR. The number of TPH-positive cells was significantly lower in mice 3 and 10 weeks after OVX than in intact WT female mice (a, d, g). LY3201 treatment significantly restored TPH-positive cells in the groups 1 week and 3 weeks after OVX (c, f). Estradiol pellets did not fully restore TPH expression in mice 3 weeks after OVX (e) although it restored the expression of TPH in 1 week (b). LY3201 treatment 10 weeks after OVX did not restore TPH-positive cells and neither did E2 treatment (h, i). Data are presented as means±s.e.m. *Po0.05 versus untreated females, Po0.05 versus placebo-treated mice in the group. Scale bar represents 100 mm.

Molecular Psychiatry (2013), 674 -- 680 & 2013 Macmillan Publishers Limited Estrogen receptor b in the dorsal raphe H Suzuki et al 677 treated group. The mice were then subdivided into three groups as primary center of serotonin projections, with known functions in follows: (1) nine mice treated with vehicle and nine with LY3201 were modulating the limbic system and thus mood disorders. submitted to a glucose tolerance test; (2) nine mice treated with vehicle and nine with LY3201 were submitted to an insulin tolerance test; and (3) Effects of gender and age on expression of TPH in the DR nine mice treated with vehicle and nine with LY3201 were used for blood To observe the effect of age and gender on serotonergic neurons, and tissue collection. we examined expression of TPH, which is the rate-limiting enzyme in the synthesis of serotonin (5-HT) in the brain. Brains of 3- and Real-time reverse transcription PCR 15-month-old WT mice were immunostained with a mono- Total RNA was extracted from frozen skeletal muscle and white adipose clonal antibody raised against TPH; n ¼ 5). As expected, TPH tissue by using the RNeasy Mini KIT (Qiagen, Valencia, CA, USA) followed by was abundantly expressed in neurons of the DR. There was no cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen, sex difference and no change with aging in the number of Grand Island, NY, USA). All genes were analyzed with the SYBR green TPH-positive neurons (Figure 2). We counted the number of TPH- detection method by using the Applied Biosystems 7500 real-time PCR positive cells in the whole DR and lateral part of the DR separately because it has been reported previously that the lateral but not machine (Foster City, CA, USA) and each PCR was plated in triplicate. 36B4 18 was used as internal control for each of the PCR ampliﬁcations. The the ventromedial DR is the stress-sensitive DR subregion. following primer sequences were used: for insulin receptor substrate-1 (IRS-1), 50-TCTACACCCGAGACGAACACT-30 and 50-TGGGCCTTTGCCCGAT Effect of inactivation of ERb on TPH in the DR TATG-30; for phosphoinositide-3-kinase (PI3K), 50-CGAGAGTGTCGTCACAG There was a marked decrease in the expression of TPH in ERbÀ/À TGTC-30 and 50-TGTTCGCTTCCACAAACACAG-30; for 36B4, 50-GCTGATGGG mice (Figure 2). In addition, many TPH-positive neurons in the CAAGAACAC-30 and 50-ATGTGAGGCAGCAGTTTCTC-30; for glucose trans- lateral part of the DR were spindle shaped. No spindle-shaped porter 4 (GLUT4), 50-CCGCGGCCTCCTATGAGATACT-30 and 50-AGGCACCCC cells were detected in the DR of aged WT mice. The data suggests GAAGATGAGT-30. that change in the morphology of the neurons of the lateral part of the DR resulted from the loss of ERb.

RESULTS The effects of OVX and treatment with E2 or ERb-selective agonist Expression of ERs in the DR of adult male mice on the expression of TPH in the DR

At the age of 6 months, the mid brains of ﬁve WT male mice were Although it is known that E2 affects serotonergic activities, the site immunostained with antibodies against ERa and ERb. Several ERa- of E2 action has been unclear because there is no expression of positive neurons were found in the periaqueductal gray but none ERa in the DR. We examined the impact of estrogen ablation on was detected in the DR. ERb-positive neurons were abundant in the serotonergic neurons and effects of E2 or ERb-selective agonist the DR (Figure 1). All staining was nuclear. These data imply that (LY3201) supplementation on this system. At the age of 6 months, ERb, but not ERa, may directly inﬂuence the DR, which is the WT female mice were ovariectomized (OVX). At 1, 3 and 10 weeks

placebo E2 LY3201 abc 0vx week 1

def Ovx 3 weeks

ghi Ovx 10 weeks

Figure 4. Photomicrographs represent estrogen receptor (ER)b-positive cells in the dorsal raphe (DR) after OVX and treatment with placebo, E2 or LY3201 for 3 days (n ¼ 5). The ERb-positive cells gradually decreased after ovariectomy (OVX) in the placebo-treated group (a, d, g). In the E2-treated group there was a small restoration of ERb-positive cells (b, e). LY3201 treatment strongly restored ERb expression in the groups in which treatment started 1 and 3 weeks (c, f) after OVX but had no effect in the group in which replacement started 10 weeks after OVX (i). Scale bars are 100 mm in lower magniﬁcation and 20 mm in higher magniﬁcation.

& 2013 Macmillan Publishers Limited Molecular Psychiatry (2013), 674 -- 680 Estrogen receptor b in the dorsal raphe H Suzuki et al 678 after the OVX, these mice were implanted with placebo, E2 or Both E2 and LY3201 supplementation increased the ERb expression LY3201 pellets subcutaneously (n ¼ 5). Three days after implanta- in the DR but a more intense effect was observed in LY3201-treated tion the mid brain was examined by immunohistochemistry. mice (Figure 4). The expression of ERb gradually decreased in the Compared with intact 6-month-old WT female mice, there was a mice treated with placebo pellets 3 weeks after the OVX, but ERb signiﬁcant decrease in the number of TPH-positive neurons in the immunoreactivity was maintained by E2 or LY3201 treatment. Ten lateral part of the DR 3 and 10 weeks after the OVX (Figure 3). In weeks after OVX there was a marked reduction in the number of the LY3201 and E2 treatment groups, when treatment was started ERb-positive neurons, and E2 and LY3201 treatment resulted in only 1 and 3 weeks after OVX, the number of TPH-positive neurons was small increases in ERb staining (Figure 4). higher than in the placebo-treated OVX mice. However, 10 weeks after estrogen deprivation, a 3-day treatment with LY3201 could no longer restore TPH-positive neurons (Figure 3). These changes were Glucose and insulin tolerance tests of the mice treated with also apparent especially in lateral part of the DR. ERb-selective agonist As we have previously reported that another ERb agonist, DPN, is diabetogenic, we examined glucose homeostasis in the mice The effects of OVX and treatment with E2 or ERb-selective agonist treated with LY3201. Before testing, the mice were fasted for 12 h on the expression of ERb in the DR and then received 1.0 U kgÀ1 of normal insulin (Humulin R, Eli Lilly) We analyzed the change of ERb-expression in the DR. One week or 1 g kgÀ1 body weight of a 100 mg mlÀ1 glucose solution in after the OVX, ERb-positive neurons were still abundant in the DR. sterile water delivered by intraperitoneal injection.

Figure 5. Comparison of glucose tolerance tests (GTTs; n ¼ 9) and insulin tolerance tests (ITTs; n ¼ 9) and their respective areas under the curve (AUC) in (wild-type) WT mice treated with vehicle (Veh) or LY3201 for 3 days. (a) GTT in WT mice treated with Veh or LY3201. No signiﬁcant differences were observed in the GTT. (b) ITT in WT mice treated with Veh or LY3201. At the times 0, 5 and 120 min after a bolus injection of insulin, the glucose levels in LY3201-treated mice were higher than in the Veh-treated mice. (c) Measurement of the AUC of GTT in WT mice treated with Veh or LY3201. (d) Measurement of the AUC of ITT in WT mice treated with Veh or LY3201. (e) Levels of insulin-regulated mRNA in skeletal muscle (SM) and white adipose tissue (WAT) of WT mice treated with Veh or LY3201 (n ¼ 9). Treatment with LY3201 for 3 days decreased the level of glucose transporter 4 (GLUT4) mRNA in SM. In WAT, treatment with LY3201 for 3 days increased the levels of GLUT4, insulin receptor substrate-1 (IRS-1) and phosphoinositide-3-kinase (PI3K) mRNA. Data are presented as mean±s.e.m. *Po0.05 versus Veh- treated mice.

Molecular Psychiatry (2013), 674 -- 680 & 2013 Macmillan Publishers Limited Estrogen receptor b in the dorsal raphe H Suzuki et al 679 No significant differences were observed in the glucose behavior of mice after OVX was restored by the treatment with 24,25 tolerance test: Thirty minutes after a bolus of glucose was given E2 or DPN, but the changes in behavior and the efficacy of E2 intraperitoneally to vehicle- and LY3201-treated mice, the glucose replacement as a function of the time after OVX have not been peak levels and the area under the plasma concentration/time studied. Such studies are required for understanding the curve (areas under the curve, AUC) in LY3201-treated mice was mechanism of postmenopausal mood disorder and the involve- slightly lower than in the vehicle-treated mice (Figures 5a and c). ment of ERb. After overnight fast, LY3201-treated mice had a small but We have previously reported that the use of DPN for 3 days significantly higher glucose level than did vehicle-treated mice reduced GLUT4 expression in skeletal muscle13 and that the block (110 vs 90 mg dlÀ1 glucose). Five and 120 min after a bolus insulin of ERb by tamoxifen in ERaÀ/À mice increased GLUT4 protein was given intraperitoneally to vehicle- and LY3201-treated mice, expression and improved glucose homeostasis.26 These results the glucose peak levels in LY3201-treated mice were higher than raised the possibility that ERb activity could have diabetogenic in the vehicle-treated mice, and there was a small increase of AUC effects. We have also reported that in ERbÀ/À mice there is a delay (Figures 5b and d). in the first phase of insulin release after an intraperitoneal bolus of glucose.26 In the present study, we observed very mild alterations in glucose homeostasis with LY3201 treatment for 3 days. Instead, Insulin signaling cascade in skeletal muscle and white adipose tissue the glucose tolerance test revealed a discrete improvement in glucose tolerance, with decreased AUC. Although the insulin Compared with vehicle-treated mice, treatment with LY3201 for 3 tolerance test indicated some decrease in insulin sensitivity, upon days caused a small reduction of GLUT4 expression and had no treatment with LY3201, the changes were within normal range. effects on IRS-1 or PI3K mRNA levels in skeletal muscle. However, In skeletal muscle, no changes were observed in IRS-1 or PI3K in white adipose tissue, LY3201 treatment caused a twofold but there was a small decrease in GLUT4 expression. However, in increase in the levels of GLUT4, IRS-1 and PI3K mRNA (Figure 5e). adipose tissue, we detected a significant increase in GLUT4, IRS-1 and PI3K mRNA. Skeletal muscle is responsible for at least 75% of insulin-induced glucose uptake,27 and recent studies in diabetic DISCUSSION patients demonstrate that a reduction of GLUT4 expression It has been pointed out before that estrogen might affect the in skeletal muscle by as little as 9% correlates with type 2 serotonin system and consequently alter mental status.19 Previous diabetes.28--30 In our study, however, the reduction in GLUT4 gene studies have demonstrated that ERb mRNA was found in the expression was not enough to induce insulin resistance. DR.11,20 The expression of ERb mRNA in the DR is reduced after We conclude that 3-day treatment with LY3201 causes only OVX and replacement with E2 can restore the gene expression mild alterations in glucose homeostasis but effectively restored profile toward normal.21 Our immunohistochemical study shows TPH expression in the DR. We envisage that, rather than the presence of ERb in the DR, which is the primary brain center of estradiol, ERb agonists could be useful pharmaceuticals in serotonin projections. The expression of ERb, not ERa, in the DR is maintaining functional neurons in the DR to treat postmenopausal enough to envisage a role for this receptor in the serotonergic depression. systems and in modulation of the limbic system and mood disorders. The number of TPH-positive cells in the DR did not change with CONFLICT OF INTEREST À/À age or gender in WT mice. However, as ERb aged, there was a V Krishnan is employed by Eli Lilly. marked reduction in the number of TPH-positive normal-looking neurons and an increase in TPH-positive spindle-shaped cells in the lateral DR. It has been reported previously that the lateral part ACKNOWLEDGEMENTS 18 of the DR is the stress-sensitive subregion of the DR and is also This study was supported by the Robert A. Welch Foundation, the Emerging 19 sensitive to E2. In addition, ERb but not ERa is expressed in the Technology Fund of Texas and the Swedish Cancer Society. Drug was provided by Eli DR of adult mice. In view of this, it is possible to infer that loss of Lilly, Indianapolis, IN, USA. ERb function will lead to adult-onset changes in mood and the lateral part of the DR may have an important role in these changes. REFERENCES One of the problems of treating depression with selective 1 Sugiyama N, Andersson S, Lathe R, Fan X, Alonso-Magdalena P, Schwend T et al. serotonin reuptake inhibitors (SSRIs) is a delay in the onset of Spatiotemporal dynamics of the expression of estrogen receptors in the postnatal 22 action of these drugs that can be as long as 6 weeks. The mode mouse brain. Mol psychiatry 2009; 14: 223--232, 117. of action of the SSRIs is elevated synaptic concentrations of 2 Fan X, Warner M, Gustafsson JA. Estrogen receptor beta expression in the serotonin (5-HT). 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