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Differential distribution and regulation of estrogen receptor-α and -β mRNA within the female rat brain1
Molecular Brain Research 54 Ž1998. 175–180
Interactive report
Differential distribution and regulation of estrogen receptor-a and -b mRNA within the female rat brain 1 a ¨ ˚ Gustafsson b, Yasmin L. Hurd Marie Osterlund , George G.J.M. Kuiper b, Jan-Ake a
a,)
Department of Clinical Neuroscience, Section of Psychiatry, Karolinska Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden b Center for Biotechnology and Department of Medical Nutrition, Karolinska Institute, Huddinge, Sweden Received 28 October 1997
Abstract In the present study, estrogen receptor ŽER. a and ERb genes were found to be differentially expressed in discrete subregions of the rat amygdaloid complex. The amygdala nuclei showing predominant ERa mRNA expression included the posterolateral cortical nucleus, amygdala hippocampal area, and lateral dorsolateral nucleus, whereas the amygdala areas with predominant ERb mRNA expression were the medial anterodorsal and central nuclei. Both ERa and ERb mRNAs were highly expressed in the medial posterodorsal nucleus. In addition to the discrete anatomical expression patterns, there appeared to be a differential regulation by estradiol of the ERa and ERb mRNAs. Two weeks of estradiol Ž170 mg total. treatment decreased ERa mRNA expression levels in the arcuate, ventromedial hypothalamus, and posterolateral cortical amygdala nucleus, but increased ERb mRNA in the arcuate. In the medial amygdala nuclei, only ERb mRNA levels were altered Žreduced. by estradiol treatment. These results suggest that estrogen can modulate behaviors and functions mediated by the amygdala and hypothalamus via differentially regulated ER subtypes. q 1998 Elsevier Science B.V. Keywords: Amygdala; Hypothalamus; 17b-estradiol; Steroid hormone; In situ hybridization
1. Introduction The estrogen receptors ŽERs. are members of the nuclear receptor superfamily. Nuclear receptors are ligandactivated transcription factors, which regulate the expression of target genes by binding to specific response elements on DNA w5,38x. In the brain, estrogen modulates neuronal activity through the ERs, but there are also reports about non-classical actions of steroids that are rapid non-genomic effects suggested to occur in the cell membrane w2,22x. Besides the influence of estrogen on hypothalamic–pituitary–gonadal functions and sexual behavior, there are several studies showing effects of estrogens on locomotion activity, mood, memory, and cognition w9,34,35x. In humans, estrogen levels seem to be of importance for the expression of psychiatric and neurological disorders such as schizophrenia, depression, and Alzheimer’s disease w11,28,36,37x. For example, estrogen is suggested to have a ‘neuroleptic-like effect’ and thereby ) Corresponding author: Tel.: Žq46-8. 517 723 79; Fax: Žq46-8. 34 65 63; E-mail: [email protected] 1 First published on the World Wide Web on 10 December 1997.
0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 3 5 1 - 3
influence the vulnerability threshold for the outbreak of schizophrenia: Schizophrenic women show an increased rate of relapse on occasions when estrogen levels are low, such as during menstruation, premenstruum, postpartum, and after menopause, whereas relapse rates are low during pregnancy, when estrogen levels are high w6,15,27x. In addition to several other gender differences in the symptomatology of schizophrenia, women have a higher mean age at onset for the disease than men Žca. 4 years. w8,11,29x. In order to elucidate the underlying mechanisms of how estrogen affects brain function and behavior, it is important to determine the distribution of ERs and the regulation of the ER genes. The discovery of a novel ER ŽERb . subtype w17x, in addition to the ERa Žthe first ER that was cloned., gives new possibilities to understand mechanisms for estrogenic effects in the brain. In situ hybridization histochemical studies of the rat brain have showed that the ERa mRNA is expressed to a great extent in the hypothalamus and the amygdala w32x. To date, only the hypothalamic distribution of the ERb mRNA has been reported w31x. The present study was designed to investigate the distributional differences between the ERa and ERb mRNA expression with an emphasis on the amygdala. In addition, because
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17b-estradiol levels fluctuate during the estrous cycle w4x and have been shown to down-regulate ERa mRNA levels in some brain areas w19,30x, we wanted to investigate if the expression of ERb mRNA is also sensitive to 17bestradiol.
2. Materials and methods Eighteen female Sprague Dawley rats Ž2.5 month old. ŽB & K Universal, Sollentuna, Sweden. were maintained on a 12 h lightrdark schedule at 198C and had free access to food pellets and water. The rats underwent bilateral ovariectomy ŽOVX. Žanesthetized with enflurane using a Tec 3 Continues Flow vaporizer — BOC Ohmeda, Vastra ¨ Frolunda, Sweden. and were allowed to recover for one ¨ week before subcutaneous insertion of pellets containing 25 or 250 mg 17b-estradiol or corresponding control placebo pellets ŽInnovative Research of America, Toledo, OH, USA.. The pellets are designed to release 17b-estradiol at steady state over a 21 day time period. The animals were killed two weeks after insertion of the pellets Žcalculated total 17b-estradiol release, 17 and 170 mg.. The brains were quickly dissected, frozen on dry ice, and stored at y708C. Sections Ž20 mm thick, coronal. were cut in a cryostat ŽJung-Frigocut 2800E cryostat, Leica., thawmounted onto glass slides Žcoated with poly-L lysine., dried at 378C, and then stored at y208C. Sera from all animals were analyzed for estradiol levels using a commercial kit ŽDPC, Los Angeles, CA, USA. with the limit of detection at 20 pgrml. Prior to in situ hybridization experiments, slides were fixed as previously described w14x. An 850 bp XbaI-EcoRI from the ERa cDNA Žprovided by Dr M. Muramatsu w16x. encoding the E and F domains and part of the 3X-UTR of rat ERa cDNA was subcloned into the EcoRV site of pBluescript KS ŽStratagene, La Jolla, CA, USA.. Antisense and sense riboprobes were generated by in vitro transcription, in the presence of 35 S-UTP ŽAmersham, Buckinghamshire, UK., with T3and T7-RNA polymerase from EcoRI and HindIII linearized plasmid respectively. A 360 bp AccI-PstI fragment encoding part of the 5X-UTR and ArB domain of rat ERb cDNA w17x was subcloned in pBluescript KS. Sense and antisense riboprobes were generated by in vitro transcription, in the presence of 35 S-UTP, with T3- and T7-RNA polymerase after plasmid linearization with PstI or AccI respectively. The labeled riboprobes were then separated from unincorporated nucleotides using microspin columns ŽS-200 HR, Pharmacia Biotech, Stockholm, Sweden.. The riboprobe hybridization was carried out as previously described w14x. In brief, 35 S-labeled probes were added to the hybridization cocktail Ž1 mgrml sheared ssDNA, 500 mgrml yeast tRNA, 2 = Denhardt’s solution, 20% dextran sulfate, 8 = sodium citrate buffer ŽSSC., and 50% formamide. to the final concentration of 20 000 cpm per ml, and 25 ml were applied to each section. The
sections were coverslipped to prevent evaporation and the hybridization was carried out in a humidified chamber overnight at 558C. After hybridization, the sections were rinsed in 2 = SSC followed by RNAse A treatment Ž40 mgrml. for 30 min at 378C. The sections were then washed in graded series of SSC, Ž2 = , 1 = , 0.5 = , 0.5 = r50% formamide, 0.1 = . containing 1 mM DTT, all at room temperature except for the formamide Ž488C. and 0.1 = SSC Ž538C. washes. Subsequently, the sections were dehydrated with ethanol containing 300 mM ammonium acetate. The slides were then air dried and exposed to Amersham b-max Hyperfilm with 14 C standards for 2–4 weeks. Autoradiograms were scanned in at a resolution of 600 dpi with a ScanMaker II ŽMicrotek Electronics, Dussel¨ dorf, Germany.. Light transmittance values were measured from the digitalized images using Density Slice Žto subtract tissue background. in the Macintosh-based image analysis software system ŽIMAGE; Wayne Rasband, NIMH, Bethesda, MD.. The light transmittance values were converted to dpmrmg using the co-exposed standards. Coronal sections were examined at levels y1.80 to y4.16 mm relative to Bregma, in conjunction with Paxinos and Watson stereotaxic atlas w24x. Statistical evaluations were assessed by analysis of variance and Tukey– Kramer post-comparison test using the JMP Ž3.1v. statistical software package.
3. Results In situ hybridization histochemistry studies revealed distinct distribution patterns for both ERa and ERb mRNA in the female rat brain ŽFig. 1.. The expression of ERa mRNA was most dense in the medial posterodorsal amygdala ŽMePD. nucleus, amygdala hippocampal area, anterolateral ŽAHiAL., ventromedial hypothalamus ŽVMH., arcuate ŽArc., premammillary nucleus ventral ŽPMV., and posterolateral cortical amygdaloid nucleus ŽPLCo.. The expression of ERb mRNA was most dense in the paraventricular hypothalamic nucleus ŽPVN., supraoptic nucleus ŽSO., PMV, and MePD nucleus. Both ER subtype mRNAs were widely distributed throughout the amygdala, but the patterns of expression differed ŽFig. 1.. The medial amygdala showed the most heterogeneous expression pattern of the regions examined. ERa and ERb mRNAs were both expressed in low amounts within the ventral medial nuclei. Moderate levels of the b-subtype mRNA were observed throughout the extent of the medial anterodorsal ŽMeAD. nucleus, whereas the ERa mRNA expression was predominantly within the most medial aspects ŽFig. 1B.. The ER mRNAs were abundantly expressed in the MePD: ERb had a more intense hybridization signal than the ERa , but localized within a more restricted area of the MePD ŽFig. 1C.. In the AHiAL, ERa mRNA expression was intense, whereas the
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Fig. 1. Distribution of ERa and ERb mRNA within the female ovariectomized rat brain throughout the rostral-to-caudal extent of amygdala and hypothalamus ŽA–E.. AHiAL, amygdala hippocampal area, anterolateral; Arc, arcuate nucleus; BM, basomedial amygdala nucleus; CA3, field of hippocampus; Ce, Central amygdala nucleus; DEn, dorsal endopiriform nucleus; LaDL, lateral dorsolateral amygdala nucleus; LH, lateral hypothalamic area; MeAD, medial anterodorsal amygdala nucleus; MePD, medial posterodorsal amygdala nucleus; MTu, medial tuberal nucleus; Pe, periventricular hypothalamic nucleus; Pir, piriform cortex; PVN, paraventricular hypothalamic nucleus; PLCo, posterolateral cortical amygdala nucleus; PMV, premammillary nucleus ventral; SO, supraoptic nucleus; VMHDM, ventromedial hypothalamus dorsomedial; VMHVL, ventromedial hypothalamus ventrolateral.
ERb mRNA was undetectable ŽFig. 1D.. Neither of the two subtypes were present in the posteromedial nucleus of the AHiA. ERb mRNA was moderately expressed in the central amygdala ŽCe. nucleus where the hybridization
signal was barely detectable for the a-subtype ŽFig. 1A and B.. In the basomedial amygdala ŽBM. nuclei, ERa mRNA was low in abundance and ERb mRNA expressed in very low levels ŽFig. 1A.. The lateral dorsolateral
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amygdala ŽLaDL. also expressed low levels of ERa mRNA, increasing slightly throughout the rostral-to-caudal extent of the nucleus ŽFig. 1B–D.. No positive ERb mRNA hybridization signal was present in the LaDL. In the Co nuclei, the mRNA of the b-subtype was not detectable. However, the ERa mRNA was expressed at high levels within the PLCo, but at very low levels in the posteromedial and anterior Co nuclei ŽFig. 1B–D.. As described by Shugrue et al.w31x, the major difference in hypothalamic distribution found between ERa and ERb mRNA was the dense presence of ERb within the PVN and SO nucleus, that contained little or no ERa mRNA, whereas the Arc and the VMH ventrolateral showed much higher expression of the a- compared to the b-subtype mRNA ŽFig. 1.. Both ERa and ERb mRNAs were highly expressed in the PMV. The periventricular, medial tuberal, and lateral hypothalamic nuclei expressed only ERa mRNA in low-to-moderate amounts ŽFig. 1A, C and D.. Both mRNA subtypes were expressed in the cerebral cortex: the ERb was more heavily expressed in layer V and no distinct cortical lamination was evident for ERa Ždata not shown.. The two subtypes were moderately expressed in the piriform cortex, while the a-subtype alone was expressed Žlowly. within the dorsal endopiriform cortex ŽFig. 1.. In addition to the above-mentioned brain areas, low-to-moderate positive hybridization signals were apparent in the zona inserta ŽFig. 1B., habenula Ždata not shown., and hippocampal formation Žmore intense in ventral CA3. for both ERa and ERb mRNAs ŽFig. 1E.. Aside from the distinct hybridization patterns observed using the a and b antisense riboprobes, the specificity of the hybridization signals was also validated with the sense probes. No positive hybridization signals were found in most brain areas studied with the ERa or ERb sense riboprobes, except for weak levels in the cerebral cortex Žin particular with the ERa sense probe., hippocampal formation, and thalamus Ždata not shown..ERa and ERb mRNA expression levels were found to be influenced by 17b-estradiol treatment within the amygdala and hypothalamus as shown in Fig. 2. Two weeks of 17b-estradiol treatment following OVX reduced the levels of ERa mRNA in the Arc, VMH, and PLCo. The highest 17bestradiol dose, 170 mg Žresulting in serum levels of approximately 130 pgrml, that is just above the physiological levels during proestrus w4x., reduced the ERa mRNA expression within the Arc, VMH, and PLCo by 33% Ž p - 0.02., 36% Ž p - 0.04., and 26% Ž p - 0.001. compared to the placebo treatment, respectively. There were no significant changes in ERa mRNA expression within the MeAD, MePD, AHiAL, LaDL, and BM nuclei. The lower 17b-estradiol dose, 17 mg Žresulting in serum levels below 20 pgrml, consistent to physiological levels maintained during late estrus and early metestrus w4x., did not change ERa mRNA levels significantly in any of the examined regions. Inversely, the expression of ERb mRNA levels was increased Ž12%; p - 0.01, compared to placebo.
Fig. 2. Influence of 17b-estradiol treatment on ERa ŽA. and ERb ŽB. mRNA levels within the amygdala and hypothalamus. Three groups Ž ns6. of female ovariectomized rats were implanted Žs.c.. with pellets releasing 17 or 170 mg 17b-estradiol or control placebo over a 14 day period. In situ hybridization signals were quantified as dpmrmg and adjusted as percentage of placebo. Data are reported as the mean "SEM. ), P - 0.05; )), P - 0.01; ))), P - 0.001, compared to placebo. Abbreviations, see Fig. 1.
within the Arc nucleus after 17b-estradiol treatment Ž170 mg., but decreased in the MeAD and MePD nucleus Ž6%; p - 0.001 and 20%; p - 0.03, respectively.. The lower 17b-estradiol dose also reduced ERb mRNA levels by 5% Ž p - 0.001. in the MeAD. Expression of ERb mRNA levels remained constant in the Ce nucleus and VMH.
4. Discussion The current in situ hybridization results confirm and extend previous findings regarding the distribution of ERa and ERb mRNA in the rat brain w31,32x. In addition to the previous distributional differences reported between the two ER mRNA subtypes within the hypothalamus w31x, this study demonstrates clearly that the two ER genes have distinct expression patterns within the amygdaloid complex. Although the hybridization signals from the ERa and ERb riboprobes cannot be compared quantitatively, the differential distributional patterns do not appear to be related to the different sizes of the two riboprobes because
¨ M. Osterlund et al.r Molecular Brain Research 54 (1998) 175–180
the shorter ERb probe produced much more intense hybridization signals than the ERa probe in specific areas, e.g., the PVN and MePD nuclei. The a-subtype was solely expressed within the AHiAL, LaDL, and all Co amygdala nuclei, but was barely detectable in the Ce nucleus where the ERb mRNA was expressed in low to moderate amounts. Moreover, when both subtypes were present within the same nuclei, e.g. the MeAD and MePD, they showed different subregional expression patterns suggesting different phenotypes of ERa and ERb mRNA-expressing cell populations. Although most studies have focused on estrogen regulation of neuroendocrine function in the hypothalamus, our findings indicate a wide expression of ER transcripts throughout the amygdaloid complex consistent with the presence of estradiol binding sites w26x and ERa immunoreactivity w20x. The current mRNA expression pattern for the ERa is similar to the pattern previously described by Simerly et al. w32x. However, moderate ERa mRNA expression noted in their summery table for the Ce nucleus does not correlate with the corresponding figure that more closely resembles the present findings showing sparse, if any, ERa mRNA expression in the Ce. ERa-immunoreactivity neurons have been found in the Ce nucleus of the female hamster, but just scattered to the medial edge of this nuclei w20x. In a recent immunohistochemical study of the female rat brain w21x, the anatomical pattern of ERb immunoreactivity was generally found to be in agreement with the areas expressing high or intense ERb mRNA in the present study. However, the MePD amygdala nucleus, that had intense ERb mRNA expression, showed no ER-b immunoreactivity. There was also a discrepancy in the areas showing low to moderate mRNA expression of the ERb because these regions had no ER-b containing cells. These differences may be due to the sensitivity of the immunohistochemical method to detect low ERb-expressing neurons. Functionally, the amygdaloid complex is considered a critical anatomical site for the control of emotions. The Ce nucleus Žthat had relatively higher expression of ERb mRNA. is the major intra-amygdaloid projection target and it has been strongly linked to agonistic behavior w7x. A substantial output from the amygdala to the VMH, that for example regulates sexual behavior w25x, originates from the AHiA, medial, and cortical nuclei w1x, areas with intense expression of ERa mRNA. The AHiA, an area with predominant expression of the ERa mRNA, provides a major route through which amygdala nuclei reach the hippocampus Žkey anatomical site for learning and memory.. The medial nucleus, that contained both ER subtypes, is also associated with defensive behavior, and in humans, amygdala lesions within the medial nucleus have been shown to blunt emotional response w23x. Overall, the wide expression of the ERa and ERb mRNAs within the amygdaloid complex suggests that estrogen should be able to influence a number of emotional behaviors related to
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fear, anxiety, sexuality, aggression, cognition, associative learning, and formation of long-term memory. In addition to the differential distribution of the ERa and ERb mRNA transcripts, we demonstrate for the first time a region-specific differential regulation of the ER mRNA subtypes by estradiol. The reduction of ERa mRNA observed within the VMH, Arc, and PLCo, but no alteration within the medial amygdala after 17b-estradiol treatment, is in agreement with many other studies w18,19,33x. The estradiol sensitivity of the ERb mRNA within the rat brain is a novel finding. Interestingly, despite lower overall expression of the ERb mRNA, the b subtype was increased after estradiol treatment within the Arc in contrast to the reduced ERa mRNA, even though the decrease in a-subtype levels was of a greater magnitude than the increase of ERb. Estrogen treatment has been shown to reduce estradiol binding sites in the medial amygdala w3x. Our results would suggest that this reduction might relate more to the b-subtype since ERa mRNA was unchanged in the medial amygdala Žcurrent results, w18x., whereas ERb mRNA levels were decreased. One explanation for the mechanisms involved in the differential region-specific estradiol regulation of both the a and b mRNA transcripts could be alternative promoter usage. Multiple promoters, that can be differentially regulated and are under cell-specific control, are present in both the rat and human ERa gene w10,12,13x. The fact that two different subtypes of ERs are differently regulated and show distinct neuroanatomical patterns strongly suggest that they play different roles in regulating brain function and behavior. Acknowledgements
¨ We thank Ms. Haleh Razani and Prof. Sven-Ove Ogren for generously sharing technical services with handling the animals and Ms. Siv Eriksson for technical assistance. This work was supported by grants from the Stiftelsen Sode¨ rstrom Sjukhemmet, Fredrik och Ingrid Thurings ¨ Konigska ¨ Stiftelse, and Kapten Arthur Erikssons Stiftelse. References w1x D.G. Amaral, J.L. Price, A. Pitkanen, S.T. Carmichael, Anatomical ¨ organization of the primate amygdaloid complex, in: J.P. Aggleton ŽEd.., The Amygdala: Neurobiological aspects of emotion, memory, and mental dysfunction, Wiley-Liss, New York, 1992, pp. 1–66. w2x D.W. Brann, L.B. Hendry, V.B. Mahesh, Emerging diversities in the mechanism of action of steroid hormones, J. Steroid Biochem. Mol. Biol. 52 Ž1995. 113–133. w3x T.J. Brown, S. Bernadette, R.B. Hochberg, N.J. MacLusky, Regulation of estrogen receptor concentrations in the rat brain: Effects of sustained androgen and estrogen exposure, Neuroendocrinology 63 Ž1996. 53–60. w4x R.L. Butcher, W.E. Collins, N.W. Fugo, Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17b throughout the 4-Day estrous cycle of the rat, Endocrinology 94 Ž1974. 1704–1708.
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w5x M.-A. Carson-Jurica, W.T. Schrader, B.W. O’Malley, Steroid receptor family: Structure and functions, Endocrine Rev. 11 Ž1990. 201–220. w6x S.S. Chang, D.C. Renshaw, Psychosis and pregnancy, Compr. Ther. 12 Ž1986. 36–41. w7x M. Davis, D. Rannie, M. Cassel, Neurotransmission in the rat amygdala related to fear and anxiety, TINS 17 Ž1994. 208–214. w8x S.V. Faraone, W.J. Chen, J.M. Goldstein, M.T. Tsuang, Gender differences in age at onset of schizophrenia, Brit. J. Psychiat. 164 Ž1994. 625–629. w9x G. Fink, B.E.H. Sumner, R. Rosie, O. Grace, J.P. Quinn, Estrogen control of central neurotransmission: Effect on mood, mental state and memory, Cell. Mol. Neurobiol. 16 Ž1996. 325–344. w10x B. Freyschuss, K. Grandien, The 5X flank of the rat estrogen receptor gene: Structural characterization and evidence for tissue- and species-specific promotor utilization, J. Mol. Endocrinol. 17 Ž1996. 197–206. w11x J.M. Goldstein, B.G. Link, Gender and the expression of schizophrenia, J. Psychiat. Res. 22 Ž1988. 141–155. w12x K. Grandien, Determination of transcription start sites in the human estrogen receptor gene and identification of a novel, species-specific, estrogen receptor-mRNA isoform, Mol. Cell. Endocrinol. 116 Ž1996. 207–212. ¨ Ljunggren, J.-A.B. ˚ Gustafsson, Estrow13x K. Grandien, M. Backdahl, O. ¨ gen target tissue determines alternative promotor utilization of the human estrogen receptor gene in osteoblast and tumor cell lines, Endocrinology 136 Ž1995. 2223–2229. w14x Y.L. Hurd, M. Herkenham, Neuropeptide compartmentalization within the human neostriatum: In situ hybridization and in vitro autoradiography characterization, Neuroscience 64 Ž1995. 571–586. w15x R.E. Kendell, J.C. Chalmers, C. Platz, Epidemiology and puerperal psychoses, Brit. J. Psychiatry 150 Ž1987. 662–673. w16x S. Koike, M. Sakai, M. Muramatso, Molecular cloning and characterization of rat estrogen receptor cDNA, Nucleic Acids Res. 15 Ž1987. 2499–2513. w17x G.G. Kuiper, E. Enmark, M. Pelto-Huikko, S. Nilsson, J.A. Gustafsson, Cloning of a novel estrogen receptor expressed in rat prostate and ovary, Proc. Nat. Acad. Sci. USA 93 Ž1996. 5925–5930. w18x A.H. Lauber, C.V. Mobbs, M. Muramatsu, D.W. Pfaff, Estrogen receptor messenger RNA expression in rat hypothalamus as a function of genetic sex and estrogen dose, Endocrinology 129 Ž1991. 3180–3186. w19x A.H. Lauber, G.J. Romano, C.V. Mobbs, D.W. Pfaff, Estradiol regulation of estrogen receptor messenger ribonucleic acid in rat mediobasal hypothalamus: An in situ hybridisation study, J. Neuroendocrin. 2 Ž1990. 605–611. w20x H.-Y. Li, J.D. Blaustein, G.J. De Vries, G.N. Wade, Estrogen-receptor immunoreactivity in hamster brain: preoptic area, hypothalamus and amygdala, Brain Res. 631 Ž1993. 304–312. w21x X. Li, P.E. Schwartz, E.F. Rissman, Distribution of estrogen receptor-b-like immunoreactivity in rat forebrain, Neuroendocrin. 66 Ž1997. 63–67.
w22x B. McEwen, Steroids affect neuronal activity by acting on the membrane and the genome, Trends. Pharmacol. Sci. 12 Ž1991. 141–147. w23x H. Narabayashi, F. Shima, Which is the better amygdala target, the medial or lateral nucleus for behavioral problems and paroxysms in epileptics, in: L.V. Laitinen, K.E. Livingston ŽEds.., Surgical Approaches in Psychiatry, University Park Press, Baltimore, 1973, pp. 129–134. w24x G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, USA, 1997. w25x D. Pfaff, Estrogen and brain function, Springer, Berlin, 1980. w26x D. Pfaff, M. Keiner, Atlas of estradiol-concentrating cells in the central nervous system of the female rat, J. Comp. Neurol. 151 Ž1973. 121–158. w27x A. Reicher-Rossler, H. Hafner, K. Maurer, M. Stummbaum, R. ¨ ¨ Schmidt, Schizophrenic symptomatology varies with serum estradiol levels during menstrual cycle, Schizophrenia Res. 6 Ž1992. 114–115. w28x A. Riecher-Rossler, H. Hafner, Schizophrenia and oestrogen — is ¨ ¨ there an association?, Eur. Arch. Psychiatry Clin. Neurosci. 242 Ž1993. 323–328. w29x M.V. Seeman, M. Lang, The role of estrogen in gender differences, Schizophr. Bull. 16 Ž1990. 185–194. w30x P.J. Shughrue, C.D. Bushnell, D.M. Dorsa, Estrogen receptor messenger ribonucleic acid in female rat brain during the estrous cycle: A comparison with ovariectomized females and intact males, Endocrinology 131 Ž1992. 381–388. w31x P.J. Shughrue, B. Komm, I. Merchenthaler, The distribution of estrogen receptor-b mRNA in the rat hypothalamus, Steroids 61 Ž1996. 678–681. w32x R.B. Simerly, C. Chang, M. Muramatsu, L.W. Swansson, Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: An in situ hybridization study, J. Comp. Neurol. 294 Ž1990. 76–95. w33x R.B. Simerly, B.J. Young, Regulation of estrogen receptor messenger ribonucleic acid in rat hypothalamus by sex steroid hormones, Mol. Endocrinol. 5 Ž1991. 424–432. w34x S.S. Smith, Female sex steroid hormones: from receptors to networks to performance — actions on the sensorimotor system, Progress in Neurobiology 44 Ž1994. 55–86. w35x S.S. Smith, Hormones, mood, neurobiology — a summary, in: G. Berg, M. Hammar ŽEds.., The modern management of the menopause. A perspective for the 21st century, Parthenon, London, 1994. w36x M.-X. Tang, D. Jacobs, Y. Stern, K. Marder, P. Schofield, B. Gurland, H. Andrews, R. Mayeux, Effect of oestrogen during menopause on risk and age at onset of alzheimer’s disease, The Lancet 348 Ž1996. 429–432. w37x S.D. Targum, K.P. Caputo, S.K. Ball, Menstrual cycle phase and psychiatric admissions, J. Affective Disorders 22 Ž1991. 49–53. w38x M.J. Tsai, B.W. Omalley, Molecular mechanisms of action of steroidrthyroid receptor superfamily members, Ann. Rev. Biochem. 63 Ž1994. 451–486.
Interactive report
Differential distribution and regulation of estrogen receptor-a and -b mRNA within the female rat brain 1 a ¨ ˚ Gustafsson b, Yasmin L. Hurd Marie Osterlund , George G.J.M. Kuiper b, Jan-Ake a
a,)
Department of Clinical Neuroscience, Section of Psychiatry, Karolinska Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden b Center for Biotechnology and Department of Medical Nutrition, Karolinska Institute, Huddinge, Sweden Received 28 October 1997
Abstract In the present study, estrogen receptor ŽER. a and ERb genes were found to be differentially expressed in discrete subregions of the rat amygdaloid complex. The amygdala nuclei showing predominant ERa mRNA expression included the posterolateral cortical nucleus, amygdala hippocampal area, and lateral dorsolateral nucleus, whereas the amygdala areas with predominant ERb mRNA expression were the medial anterodorsal and central nuclei. Both ERa and ERb mRNAs were highly expressed in the medial posterodorsal nucleus. In addition to the discrete anatomical expression patterns, there appeared to be a differential regulation by estradiol of the ERa and ERb mRNAs. Two weeks of estradiol Ž170 mg total. treatment decreased ERa mRNA expression levels in the arcuate, ventromedial hypothalamus, and posterolateral cortical amygdala nucleus, but increased ERb mRNA in the arcuate. In the medial amygdala nuclei, only ERb mRNA levels were altered Žreduced. by estradiol treatment. These results suggest that estrogen can modulate behaviors and functions mediated by the amygdala and hypothalamus via differentially regulated ER subtypes. q 1998 Elsevier Science B.V. Keywords: Amygdala; Hypothalamus; 17b-estradiol; Steroid hormone; In situ hybridization
1. Introduction The estrogen receptors ŽERs. are members of the nuclear receptor superfamily. Nuclear receptors are ligandactivated transcription factors, which regulate the expression of target genes by binding to specific response elements on DNA w5,38x. In the brain, estrogen modulates neuronal activity through the ERs, but there are also reports about non-classical actions of steroids that are rapid non-genomic effects suggested to occur in the cell membrane w2,22x. Besides the influence of estrogen on hypothalamic–pituitary–gonadal functions and sexual behavior, there are several studies showing effects of estrogens on locomotion activity, mood, memory, and cognition w9,34,35x. In humans, estrogen levels seem to be of importance for the expression of psychiatric and neurological disorders such as schizophrenia, depression, and Alzheimer’s disease w11,28,36,37x. For example, estrogen is suggested to have a ‘neuroleptic-like effect’ and thereby ) Corresponding author: Tel.: Žq46-8. 517 723 79; Fax: Žq46-8. 34 65 63; E-mail: [email protected] 1 First published on the World Wide Web on 10 December 1997.
0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 3 5 1 - 3
influence the vulnerability threshold for the outbreak of schizophrenia: Schizophrenic women show an increased rate of relapse on occasions when estrogen levels are low, such as during menstruation, premenstruum, postpartum, and after menopause, whereas relapse rates are low during pregnancy, when estrogen levels are high w6,15,27x. In addition to several other gender differences in the symptomatology of schizophrenia, women have a higher mean age at onset for the disease than men Žca. 4 years. w8,11,29x. In order to elucidate the underlying mechanisms of how estrogen affects brain function and behavior, it is important to determine the distribution of ERs and the regulation of the ER genes. The discovery of a novel ER ŽERb . subtype w17x, in addition to the ERa Žthe first ER that was cloned., gives new possibilities to understand mechanisms for estrogenic effects in the brain. In situ hybridization histochemical studies of the rat brain have showed that the ERa mRNA is expressed to a great extent in the hypothalamus and the amygdala w32x. To date, only the hypothalamic distribution of the ERb mRNA has been reported w31x. The present study was designed to investigate the distributional differences between the ERa and ERb mRNA expression with an emphasis on the amygdala. In addition, because
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17b-estradiol levels fluctuate during the estrous cycle w4x and have been shown to down-regulate ERa mRNA levels in some brain areas w19,30x, we wanted to investigate if the expression of ERb mRNA is also sensitive to 17bestradiol.
2. Materials and methods Eighteen female Sprague Dawley rats Ž2.5 month old. ŽB & K Universal, Sollentuna, Sweden. were maintained on a 12 h lightrdark schedule at 198C and had free access to food pellets and water. The rats underwent bilateral ovariectomy ŽOVX. Žanesthetized with enflurane using a Tec 3 Continues Flow vaporizer — BOC Ohmeda, Vastra ¨ Frolunda, Sweden. and were allowed to recover for one ¨ week before subcutaneous insertion of pellets containing 25 or 250 mg 17b-estradiol or corresponding control placebo pellets ŽInnovative Research of America, Toledo, OH, USA.. The pellets are designed to release 17b-estradiol at steady state over a 21 day time period. The animals were killed two weeks after insertion of the pellets Žcalculated total 17b-estradiol release, 17 and 170 mg.. The brains were quickly dissected, frozen on dry ice, and stored at y708C. Sections Ž20 mm thick, coronal. were cut in a cryostat ŽJung-Frigocut 2800E cryostat, Leica., thawmounted onto glass slides Žcoated with poly-L lysine., dried at 378C, and then stored at y208C. Sera from all animals were analyzed for estradiol levels using a commercial kit ŽDPC, Los Angeles, CA, USA. with the limit of detection at 20 pgrml. Prior to in situ hybridization experiments, slides were fixed as previously described w14x. An 850 bp XbaI-EcoRI from the ERa cDNA Žprovided by Dr M. Muramatsu w16x. encoding the E and F domains and part of the 3X-UTR of rat ERa cDNA was subcloned into the EcoRV site of pBluescript KS ŽStratagene, La Jolla, CA, USA.. Antisense and sense riboprobes were generated by in vitro transcription, in the presence of 35 S-UTP ŽAmersham, Buckinghamshire, UK., with T3and T7-RNA polymerase from EcoRI and HindIII linearized plasmid respectively. A 360 bp AccI-PstI fragment encoding part of the 5X-UTR and ArB domain of rat ERb cDNA w17x was subcloned in pBluescript KS. Sense and antisense riboprobes were generated by in vitro transcription, in the presence of 35 S-UTP, with T3- and T7-RNA polymerase after plasmid linearization with PstI or AccI respectively. The labeled riboprobes were then separated from unincorporated nucleotides using microspin columns ŽS-200 HR, Pharmacia Biotech, Stockholm, Sweden.. The riboprobe hybridization was carried out as previously described w14x. In brief, 35 S-labeled probes were added to the hybridization cocktail Ž1 mgrml sheared ssDNA, 500 mgrml yeast tRNA, 2 = Denhardt’s solution, 20% dextran sulfate, 8 = sodium citrate buffer ŽSSC., and 50% formamide. to the final concentration of 20 000 cpm per ml, and 25 ml were applied to each section. The
sections were coverslipped to prevent evaporation and the hybridization was carried out in a humidified chamber overnight at 558C. After hybridization, the sections were rinsed in 2 = SSC followed by RNAse A treatment Ž40 mgrml. for 30 min at 378C. The sections were then washed in graded series of SSC, Ž2 = , 1 = , 0.5 = , 0.5 = r50% formamide, 0.1 = . containing 1 mM DTT, all at room temperature except for the formamide Ž488C. and 0.1 = SSC Ž538C. washes. Subsequently, the sections were dehydrated with ethanol containing 300 mM ammonium acetate. The slides were then air dried and exposed to Amersham b-max Hyperfilm with 14 C standards for 2–4 weeks. Autoradiograms were scanned in at a resolution of 600 dpi with a ScanMaker II ŽMicrotek Electronics, Dussel¨ dorf, Germany.. Light transmittance values were measured from the digitalized images using Density Slice Žto subtract tissue background. in the Macintosh-based image analysis software system ŽIMAGE; Wayne Rasband, NIMH, Bethesda, MD.. The light transmittance values were converted to dpmrmg using the co-exposed standards. Coronal sections were examined at levels y1.80 to y4.16 mm relative to Bregma, in conjunction with Paxinos and Watson stereotaxic atlas w24x. Statistical evaluations were assessed by analysis of variance and Tukey– Kramer post-comparison test using the JMP Ž3.1v. statistical software package.
3. Results In situ hybridization histochemistry studies revealed distinct distribution patterns for both ERa and ERb mRNA in the female rat brain ŽFig. 1.. The expression of ERa mRNA was most dense in the medial posterodorsal amygdala ŽMePD. nucleus, amygdala hippocampal area, anterolateral ŽAHiAL., ventromedial hypothalamus ŽVMH., arcuate ŽArc., premammillary nucleus ventral ŽPMV., and posterolateral cortical amygdaloid nucleus ŽPLCo.. The expression of ERb mRNA was most dense in the paraventricular hypothalamic nucleus ŽPVN., supraoptic nucleus ŽSO., PMV, and MePD nucleus. Both ER subtype mRNAs were widely distributed throughout the amygdala, but the patterns of expression differed ŽFig. 1.. The medial amygdala showed the most heterogeneous expression pattern of the regions examined. ERa and ERb mRNAs were both expressed in low amounts within the ventral medial nuclei. Moderate levels of the b-subtype mRNA were observed throughout the extent of the medial anterodorsal ŽMeAD. nucleus, whereas the ERa mRNA expression was predominantly within the most medial aspects ŽFig. 1B.. The ER mRNAs were abundantly expressed in the MePD: ERb had a more intense hybridization signal than the ERa , but localized within a more restricted area of the MePD ŽFig. 1C.. In the AHiAL, ERa mRNA expression was intense, whereas the
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Fig. 1. Distribution of ERa and ERb mRNA within the female ovariectomized rat brain throughout the rostral-to-caudal extent of amygdala and hypothalamus ŽA–E.. AHiAL, amygdala hippocampal area, anterolateral; Arc, arcuate nucleus; BM, basomedial amygdala nucleus; CA3, field of hippocampus; Ce, Central amygdala nucleus; DEn, dorsal endopiriform nucleus; LaDL, lateral dorsolateral amygdala nucleus; LH, lateral hypothalamic area; MeAD, medial anterodorsal amygdala nucleus; MePD, medial posterodorsal amygdala nucleus; MTu, medial tuberal nucleus; Pe, periventricular hypothalamic nucleus; Pir, piriform cortex; PVN, paraventricular hypothalamic nucleus; PLCo, posterolateral cortical amygdala nucleus; PMV, premammillary nucleus ventral; SO, supraoptic nucleus; VMHDM, ventromedial hypothalamus dorsomedial; VMHVL, ventromedial hypothalamus ventrolateral.
ERb mRNA was undetectable ŽFig. 1D.. Neither of the two subtypes were present in the posteromedial nucleus of the AHiA. ERb mRNA was moderately expressed in the central amygdala ŽCe. nucleus where the hybridization
signal was barely detectable for the a-subtype ŽFig. 1A and B.. In the basomedial amygdala ŽBM. nuclei, ERa mRNA was low in abundance and ERb mRNA expressed in very low levels ŽFig. 1A.. The lateral dorsolateral
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amygdala ŽLaDL. also expressed low levels of ERa mRNA, increasing slightly throughout the rostral-to-caudal extent of the nucleus ŽFig. 1B–D.. No positive ERb mRNA hybridization signal was present in the LaDL. In the Co nuclei, the mRNA of the b-subtype was not detectable. However, the ERa mRNA was expressed at high levels within the PLCo, but at very low levels in the posteromedial and anterior Co nuclei ŽFig. 1B–D.. As described by Shugrue et al.w31x, the major difference in hypothalamic distribution found between ERa and ERb mRNA was the dense presence of ERb within the PVN and SO nucleus, that contained little or no ERa mRNA, whereas the Arc and the VMH ventrolateral showed much higher expression of the a- compared to the b-subtype mRNA ŽFig. 1.. Both ERa and ERb mRNAs were highly expressed in the PMV. The periventricular, medial tuberal, and lateral hypothalamic nuclei expressed only ERa mRNA in low-to-moderate amounts ŽFig. 1A, C and D.. Both mRNA subtypes were expressed in the cerebral cortex: the ERb was more heavily expressed in layer V and no distinct cortical lamination was evident for ERa Ždata not shown.. The two subtypes were moderately expressed in the piriform cortex, while the a-subtype alone was expressed Žlowly. within the dorsal endopiriform cortex ŽFig. 1.. In addition to the above-mentioned brain areas, low-to-moderate positive hybridization signals were apparent in the zona inserta ŽFig. 1B., habenula Ždata not shown., and hippocampal formation Žmore intense in ventral CA3. for both ERa and ERb mRNAs ŽFig. 1E.. Aside from the distinct hybridization patterns observed using the a and b antisense riboprobes, the specificity of the hybridization signals was also validated with the sense probes. No positive hybridization signals were found in most brain areas studied with the ERa or ERb sense riboprobes, except for weak levels in the cerebral cortex Žin particular with the ERa sense probe., hippocampal formation, and thalamus Ždata not shown..ERa and ERb mRNA expression levels were found to be influenced by 17b-estradiol treatment within the amygdala and hypothalamus as shown in Fig. 2. Two weeks of 17b-estradiol treatment following OVX reduced the levels of ERa mRNA in the Arc, VMH, and PLCo. The highest 17bestradiol dose, 170 mg Žresulting in serum levels of approximately 130 pgrml, that is just above the physiological levels during proestrus w4x., reduced the ERa mRNA expression within the Arc, VMH, and PLCo by 33% Ž p - 0.02., 36% Ž p - 0.04., and 26% Ž p - 0.001. compared to the placebo treatment, respectively. There were no significant changes in ERa mRNA expression within the MeAD, MePD, AHiAL, LaDL, and BM nuclei. The lower 17b-estradiol dose, 17 mg Žresulting in serum levels below 20 pgrml, consistent to physiological levels maintained during late estrus and early metestrus w4x., did not change ERa mRNA levels significantly in any of the examined regions. Inversely, the expression of ERb mRNA levels was increased Ž12%; p - 0.01, compared to placebo.
Fig. 2. Influence of 17b-estradiol treatment on ERa ŽA. and ERb ŽB. mRNA levels within the amygdala and hypothalamus. Three groups Ž ns6. of female ovariectomized rats were implanted Žs.c.. with pellets releasing 17 or 170 mg 17b-estradiol or control placebo over a 14 day period. In situ hybridization signals were quantified as dpmrmg and adjusted as percentage of placebo. Data are reported as the mean "SEM. ), P - 0.05; )), P - 0.01; ))), P - 0.001, compared to placebo. Abbreviations, see Fig. 1.
within the Arc nucleus after 17b-estradiol treatment Ž170 mg., but decreased in the MeAD and MePD nucleus Ž6%; p - 0.001 and 20%; p - 0.03, respectively.. The lower 17b-estradiol dose also reduced ERb mRNA levels by 5% Ž p - 0.001. in the MeAD. Expression of ERb mRNA levels remained constant in the Ce nucleus and VMH.
4. Discussion The current in situ hybridization results confirm and extend previous findings regarding the distribution of ERa and ERb mRNA in the rat brain w31,32x. In addition to the previous distributional differences reported between the two ER mRNA subtypes within the hypothalamus w31x, this study demonstrates clearly that the two ER genes have distinct expression patterns within the amygdaloid complex. Although the hybridization signals from the ERa and ERb riboprobes cannot be compared quantitatively, the differential distributional patterns do not appear to be related to the different sizes of the two riboprobes because
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the shorter ERb probe produced much more intense hybridization signals than the ERa probe in specific areas, e.g., the PVN and MePD nuclei. The a-subtype was solely expressed within the AHiAL, LaDL, and all Co amygdala nuclei, but was barely detectable in the Ce nucleus where the ERb mRNA was expressed in low to moderate amounts. Moreover, when both subtypes were present within the same nuclei, e.g. the MeAD and MePD, they showed different subregional expression patterns suggesting different phenotypes of ERa and ERb mRNA-expressing cell populations. Although most studies have focused on estrogen regulation of neuroendocrine function in the hypothalamus, our findings indicate a wide expression of ER transcripts throughout the amygdaloid complex consistent with the presence of estradiol binding sites w26x and ERa immunoreactivity w20x. The current mRNA expression pattern for the ERa is similar to the pattern previously described by Simerly et al. w32x. However, moderate ERa mRNA expression noted in their summery table for the Ce nucleus does not correlate with the corresponding figure that more closely resembles the present findings showing sparse, if any, ERa mRNA expression in the Ce. ERa-immunoreactivity neurons have been found in the Ce nucleus of the female hamster, but just scattered to the medial edge of this nuclei w20x. In a recent immunohistochemical study of the female rat brain w21x, the anatomical pattern of ERb immunoreactivity was generally found to be in agreement with the areas expressing high or intense ERb mRNA in the present study. However, the MePD amygdala nucleus, that had intense ERb mRNA expression, showed no ER-b immunoreactivity. There was also a discrepancy in the areas showing low to moderate mRNA expression of the ERb because these regions had no ER-b containing cells. These differences may be due to the sensitivity of the immunohistochemical method to detect low ERb-expressing neurons. Functionally, the amygdaloid complex is considered a critical anatomical site for the control of emotions. The Ce nucleus Žthat had relatively higher expression of ERb mRNA. is the major intra-amygdaloid projection target and it has been strongly linked to agonistic behavior w7x. A substantial output from the amygdala to the VMH, that for example regulates sexual behavior w25x, originates from the AHiA, medial, and cortical nuclei w1x, areas with intense expression of ERa mRNA. The AHiA, an area with predominant expression of the ERa mRNA, provides a major route through which amygdala nuclei reach the hippocampus Žkey anatomical site for learning and memory.. The medial nucleus, that contained both ER subtypes, is also associated with defensive behavior, and in humans, amygdala lesions within the medial nucleus have been shown to blunt emotional response w23x. Overall, the wide expression of the ERa and ERb mRNAs within the amygdaloid complex suggests that estrogen should be able to influence a number of emotional behaviors related to
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fear, anxiety, sexuality, aggression, cognition, associative learning, and formation of long-term memory. In addition to the differential distribution of the ERa and ERb mRNA transcripts, we demonstrate for the first time a region-specific differential regulation of the ER mRNA subtypes by estradiol. The reduction of ERa mRNA observed within the VMH, Arc, and PLCo, but no alteration within the medial amygdala after 17b-estradiol treatment, is in agreement with many other studies w18,19,33x. The estradiol sensitivity of the ERb mRNA within the rat brain is a novel finding. Interestingly, despite lower overall expression of the ERb mRNA, the b subtype was increased after estradiol treatment within the Arc in contrast to the reduced ERa mRNA, even though the decrease in a-subtype levels was of a greater magnitude than the increase of ERb. Estrogen treatment has been shown to reduce estradiol binding sites in the medial amygdala w3x. Our results would suggest that this reduction might relate more to the b-subtype since ERa mRNA was unchanged in the medial amygdala Žcurrent results, w18x., whereas ERb mRNA levels were decreased. One explanation for the mechanisms involved in the differential region-specific estradiol regulation of both the a and b mRNA transcripts could be alternative promoter usage. Multiple promoters, that can be differentially regulated and are under cell-specific control, are present in both the rat and human ERa gene w10,12,13x. The fact that two different subtypes of ERs are differently regulated and show distinct neuroanatomical patterns strongly suggest that they play different roles in regulating brain function and behavior. Acknowledgements
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