Extra-nuclear estrogen receptor GPR30 regulates serotonin function in rat hypothalamus.

Selective serotonin reuptake inhibitors (SSRIs), such as Prozac, are used to treat mood disorders. SSRIs attenuate (i.e. desensitize) serotonin 1A (5-HT(1A)) receptor signaling, as demonstrated in rats through decreased release of oxytocin and adrenocorticotropin hormone (ACTH) following 5-HT(1A) receptor stimulation. Maximal therapeutic effects of SSRIs for treatment of mood disorders, as well as effects on hypothalamic 5-HT(1A) receptor signaling in animals, take 1 to 2 weeks to develop. Estradiol also attenuates 5-HT(1A) receptor signaling, but, in rats, these effects occur within 2 days; thus, estrogens or selective estrogen receptor modulators may serve as useful short-term tools to accelerate desensitization of 5-HT(1A) receptors in response to SSRIs if candidate estrogen receptor targets in the hypothalamus are identified. We found high levels of GPR30, which has been identified recently as a pertussis-toxin (PTX) sensitive G-protein-coupled estrogen receptor, in the hypothalamic paraventricular nucleus (PVN) of rats. Double-label immunohistochemistry revealed that GPR30 co-localizes with 5-HT(1A) receptors, corticotrophin releasing factor (CRF) and oxytocin in neurons in the PVN. Pretreatment with PTX to the PVN before peripheral injections of 17-beta-estradiol 3-benzoate completely prevented the reduction of the oxytocin response to the 5-HT(1A) receptor agonist, (+)-8-hydroxy-2-dipropylaminotetralin (DPAT). Treatment with the selective GRP30 agonist, G-1, attenuated 5-HT(1A) receptor signaling in the PVN as measured by an attenuated oxytocin (by 29%) and ACTH (by 31%) response to DPAT. This study indicates that a putative extra-nuclear estrogen receptor, GPR30, may play a role in estradiol-mediated attenuation of 5-HT(1A) receptor signaling, and potentially in accelerating the effects of SSRIs in treatment of mood disorders.

Novel cellular phenotypes and subcellular sites for androgen action in the forebrain.

Historically, morphological studies of the distribution of androgen receptors in the brain led to conclusions that the major regional targets of androgen action are involved in reproduction, that the primary cellular targets are neurons, and that functional androgen receptors are exclusively nuclear, consistent with the classical view of steroid receptors as ligand-dependent transcription factors. In this review, we discuss three separate but interrelated recent studies highlighting observations made with newer methodologies while assessing the regional, cellular or subcellular distribution of androgen receptors containing cells in the forebrain. Regional studies demonstrated that the largest forebrain target for androgen action in terms of the number of androgen receptor expressing cells is the cerebral cortex, rather than the main hypothalamic and limbic centers for reproductive function. Cellular studies to determine the phenotype of androgen receptor expressing cells confirmed that most of these cells are neurons but also revealed that small subpopulations are astrocytes. The expression of androgen receptors in astrocytes is both region and age dependent. In contrast, reactive astrocytes in the lesioned adult rat brain do not express androgen receptors whereas reactive microglia do. Finally, androgen receptor immunoreactive axons were identified in the cerebral cortex of the rat and human. These observations do not overturn classical views of the cellular and subcellular locus of steroid action in the nervous system, but rather broaden our view of the potential direct impact of gonadal steroid hormones on cellular function and emphasize the regional and developmental specificity of these effects on the nervous system.

Androgen receptor expression in the developing male and female rat visual and prefrontal cortex.

Gonadal steroid hormones are known to influence the development of the cerebral cortex of mammals. Steroid hormone action involves hormone binding to cytoplasmic or nuclear receptors, followed by DNA binding and gene transcription. The goals of the present study were twofold: to determine whether androgen receptors are present during development in two known androgen sensitive regions of the rat cerebral cortex, the primary visual cortex (Oc1) and the anterior cingulate/frontal cortex (Cg1/Fr2); and to determine whether androgen receptor (AR) expression in these regions differs between developing males and females. We used immunocytochemistry to detect AR protein on postnatal days 0, 4, and 10, and in situ hybridization to detect AR mRNA on postnatal day 10 in male and female rats. The level of AR expression was specific to the cortical region, with higher AR immunoreactive cell density and more AR mRNA in Oc1 than in Cg1/Fr2. AR immunoreactive cell density increased with age in both regions. Finally, on postnatal day 10, males had a higher AR immunoreactive cell density and more AR mRNA in Oc1 than did females. Thus, the presence of ARs may allow androgens to directly influence the development the cerebral cortex.

Treatment of cycling female rats with fluoxetine induces desensitization of hypothalamic 5-HT(1A) receptors with no change in 5-HT(2A) receptors.

Although women constitute the majority of patients who receive treatment with selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, most animal studies of SSRIs are conducted on males. The present study investigated whether long-term treatment of cycling female rats with fluoxetine alters their estrous cycle and the sensitivity of hypothalamic serotonin (5-HT) 5-HT(1A) and 5-HT(2A) receptor systems. Adult female rats received daily injections of fluoxetine (10 mg/kg, i.p.) for three consecutive estrous cycles (15.2+/-0.2 days) with the first injection beginning on metestrus (when circulating estrogen levels are low and stable). Fluoxetine did not alter basal plasma estradiol levels at metestrus, nor did it alter the pattern of estrous cyclicity. Rats treated with fluoxetine showed a loss in body weight. On the morning of metestrus of the fourth cycle (18 h after the last fluoxetine injection), the rats were injected with a sub-maximal dose of the 5-HT(1A) agonist (+/-)-8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT, 50 MICRO/kg, s.c.) or a maximal dose of the 5-HT(2A) agonist [(+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl] (DOI). Plasma levels of oxytocin, ACTH and corticosterone were measured as peripheral indicators of hypothalamic 5-HT(1A) and 5-HT(2A) receptor sensitivity. Injecting 8-OH-DPAT to saline pretreated rats produced a significant increase in plasma oxytocin (299%), ACTH (1456%) and corticosterone (170%) levels but not in plasma prolactin or renin concentrations. Greater increases in plasma levels of these hormones were observed after injecting DOI. Fluoxetine treatment completely blocked the oxytocin, ACTH and corticosterone responses to 8-OH-DPAT, but did not inhibit the effect of DOI on any hormone, thus confirming that fluoxetine treatment did not produce a deficit in the functioning of corticotropin releasing hormone or oxytocin containing neurons. These results indicate that in cycling female rats, fluoxetine treatment desensitizes hypothalamic post-synaptic 5-HT(1A) receptor signaling. Understanding the pharmacological effects of fluoxetine in females may lead to more effective treatment of women with mood disorders.

Interactions of estrogens and insulin-like growth factor-I in the brain: implications for neuroprotection.

Data from epidemiological studies suggest that the decline in estrogen following menopause could increase the risk of neurodegenerative diseases. Furthermore, experimental studies on different animal models have shown that estrogen is neuroprotective. The mechanisms involved in the neuroprotective effects of estrogen are still unclear. Anti-oxidant effects, activation of different membrane-associated intracellular signaling pathways, and activation of classical nuclear estrogen receptors (ERs) could contribute to neuroprotection. Interactions with neurotrophins and other growth factors may also be important for the neuroprotective effects of estradiol. In this review we focus on the interaction between insulin-like growth factor-I (IGF-I) and estrogen signaling in the brain and on the implications of this interaction for neuroprotection. During the development of the nervous system, IGF-I promotes the differentiation and survival of specific neuronal populations. In the adult brain, IGF-I is a neuromodulator, regulates synaptic plasticity, is involved in the response of neural tissue to injury and protects neurons against different neurodegenerative stimuli. As an endocrine signal, IGF-I represents a link between the growth and reproductive axes and the interaction between estradiol and IGF-I is of particular physiological relevance for the regulation of growth, sexual maturation and adult neuroendocrine function. There are several potential points of convergence between estradiol and IGF-I receptor (IGF-IR) signaling in the brain. Estrogen activates the mitogen-activated protein kinase (MAPK) pathway and has a synergistic effect with IGF-I on the activation of Akt, a kinase downstream of phosphoinositol-3 kinase. In addition, IGF-IR is necessary for the estradiol induced expression of the anti-apoptotic molecule Bcl-2 in hypothalamic neurons. The interaction of ERs and IGF-IR in the brain may depend on interactions between neural cells expressing ERs with neural cells expressing IGF-IR, or on direct interactions of the signaling pathways of alpha and beta ERs and IGF-IR in the same cell, since most neurons expressing IGF-IR also express at least one of the ER subtypes. In addition, studies on adult ovariectomized rats given intracerebroventricular (i.c.v.) infusions with antagonists for ERs or IGF-IR or with IGF-I have shown that there is a cross-regulation of the expression of ERs and IGF-IR in the brain. The interaction of estradiol and IGF-I and their receptors may be involved in different neural events. In the developing brain, ERs and IGF-IR are interdependent in the promotion of neuronal differentiation. In the adult, ERs and IGF-IR interact in the induction of synaptic plasticity. Furthermore, both in vitro and in vivo studies have shown that there is an interaction between ERs and IGF-IR in the promotion of neuronal survival and in the response of neural tissue to injury, suggesting that a parallel activation or co-activation of ERs and IGF-IR mediates neuroprotection.

Neuroprotection by estradiol.

This review highlights recent evidence from clinical and basic science studies supporting a role for estrogen in neuroprotection. Accumulated clinical evidence suggests that estrogen exposure decreases the risk and delays the onset and progression of Alzheimer's disease and schizophrenia, and may also enhance recovery from traumatic neurological injury such as stroke. Recent basic science studies show that not only does exogenous estradiol decrease the response to various forms of insult, but the brain itself upregulates both estrogen synthesis and estrogen receptor expression at sites of injury. Thus, our view of the role of estrogen in neural function must be broadened to include not only its function in neuroendocrine regulation and reproductive behaviors, but also to include a direct protective role in response to degenerative disease or injury. Estrogen may play this protective role through several routes. Key among these are estrogen dependent alterations in cell survival, axonal sprouting, regenerative responses, enhanced synaptic transmission and enhanced neurogenesis. Some of the mechanisms underlying these effects are independent of the classically defined nuclear estrogen receptors and involve unidentified membrane receptors, direct modulation of neurotransmitter receptor function, or the known anti-oxidant activities of estrogen. Other neuroprotective effects of estrogen do depend on the classical nuclear estrogen receptor, through which estrogen alters expression of estrogen responsive genes that play a role in apoptosis, axonal regeneration, or general trophic support. Yet another possibility is that estrogen receptors in the membrane or cytoplasm alter phosphorylation cascades through direct interactions with protein kinases or that estrogen receptor signaling may converge with signaling by other trophic molecules to confer resistance to injury. Although there is clear evidence that estradiol exposure can be deleterious to some neuronal populations, the potential clinical benefits of estrogen treatment for enhancing cognitive function may outweigh the associated central and peripheral risks. Exciting and important avenues for future investigation into the protective effects of estrogen include the optimal ligand and doses that can be used clinically to confer benefit without undue risk, modulation of neurotrophin and neurotrophin receptor expression, interaction of estrogen with regulated cofactors and coactivators that couple estrogen receptors to basal transcriptional machinery, interactions of estrogen with other survival and regeneration promoting factors, potential estrogenic effects on neuronal replenishment, and modulation of phenotypic choices by neural stem cells.

Estrogen, but not androgens, regulates androgen receptor messenger ribonucleic acid expression in the developing male rat forebrain.

Testosterone is the principal gonadal hormone responsible for the masculinization of the rat nervous system. Sex differences in both the ligand and receptor availability may play a role in the process of sexual differentiation. In some brain regions, males express more androgen receptor (AR) messenger RNA (mRNA) than females by postnatal day (PND) 10. Gonadectomy on the day of birth (PND-0) eliminated the sex differences in AR mRNA expression at PND-10, and exogenous testosterone replacement restored this sex difference. Because testosterone can be converted to both androgenic and estrogenic metabolites in the brain, the present experiments were performed to determine whether androgenic or estrogenic metabolites of testosterone are responsible for region-specific regulation of AR mRNA content in the developing rat forebrain. We used a 35S-labeled riboprobe and in situ hybridization to assess relative steady-state levels of AR mRNA in animals killed on PND-10. In the principal portion of the bed nucleus of the stria terminalis (BSTpr) and medial preoptic area (MPO), males gonadectomized on PND-0 and treated daily with dihydrotestosterone propionate (DHTP), a nonaromatizable androgen, had low levels of AR mRNA that were not significantly different from AR mRNA levels in intact females. In contrast, males gonadectomized on PND-0 and treated daily with diethylstilbestrol (DES), a synthetic estrogen, maintained high, male-typical levels of AR mRNA in the BSTpr and the MPO. AR mRNA content in the VMH was not sexually differentiated in PND-10 rats and was unaffected by gonadectomy or hormone replacement. To further assess whether AR mRNA was autologously regulated, neonatal male rats were treated with the androgen receptor antagonist, flutamide. Flutamide at a dose of either 40 microg/day or 300 microg/day had no effect on AR mRNA expression in any area examined. Thus, AR mRNA is up-regulated by estrogen but is not regulated by androgen during the early postnatal period.

Differential expression of estrogen receptor alpha and beta in rat dorsal root ganglion neurons.

Estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) mRNAs are both expressed in rat dorsal root ganglion (DRG) neurons, but the distribution of these two mRNAs differs markedly. Radiolabeled probes highly specific to ERalpha or ERbeta mRNAs were used for in situ hybridization studies; two antibodies specific to ERalpha protein were used for immunocytochemistry and specific primers were used for reverse transcription polymerase chain reaction (RT-PCR) studies. These revealed that ERbeta mRNA is widely expressed in the DRG of both male and female rats, with virtually all neurons showing positive signals. In contrast, ERalpha mRNA, as well as nuclear localized ERalpha protein, is more restricted in its localization and is present in many, but not all, of the small-sized (<600 microm(2)) DRG neurons, but is only rarely present in larger neurons. The L6-S1 DRG levels, which contain sensory neurons that innervate reproductive tissues, are relatively enriched in ERalpha compared to L3-L5 DRG levels, which contain sensory neurons that innervate hind limb regions. Long-term estrogen treatment of ovariectomized rats (21-28 days) dramatically reduces immunocytochemically detectable ERalpha protein in the DRG relative to that in ovariectomized controls. RT-PCR studies also showed that long-term estrogen treatment of ovariectomized rats downregulates the levels of ERalpha mRNA, but upregulates the levels of ERbeta mRNA in the DRG. Interestingly, in intact cycling female rats, ERalpha and ERbeta mRNA levels in the DRG were both higher during proestrus compared to metestrus. These findings suggest that the changes in expression of estrogen receptors which occur dynamically during the estrus cycle differ from those induced by long-term estrogen treatment of ovariectomized animals.

Regulation of androgen receptor messenger ribonucleic acid expression in the developing rat forebrain.

By postnatal day 10 (PND-10), males express more androgen receptor (AR) messenger RNA (mRNA) than females in the principal portion of the bed nucleus of the stria terminalis (BSTpr) and medial preoptic area (MPO), but not in the ventromedial hypothalamus. The development of these region-specific sex differences in AR mRNA expression may be critical for the organization of male-typical neural circuitry and may represent the onset of sex differences in the sensitivity of the rat brain to the actions of androgens. In this study, we used a 35S-labeled riboprobe and in situ hybridization to address whether postnatal testosterone exposure is important for the up-regulation of AR mRNA content in the developing rat forebrain. In the BSTpr and the MPO of PND-10 rats, males gonadectomized on PND-0 or PND-5 had lower levels of AR mRNA compared with intact or sham-operated control males. Daily replacement of testosterone to animals gonadectomized on PND-0 maintained AR mRNA content in the BSTpr and the MPO at levels equal to those in intact males. In contrast, there was no effect of gonadectomy or testosterone replacement on AR mRNA expression in the ventromedial hypothalamus. Thus, the postnatal hormonal environment may permit the development of region-specific sex differences in AR mRNA. Significant alterations in AR mRNA expression in the BSTpr and MPO in PND-10 male rats were induced by gonadectomy as late as PND-8. Males gonadectomized on PND-8 had levels of AR mRNA significantly lower than those in intact males, but significantly higher than those in intact females. Further, when animals were gonadectomized on PND-0 and given testosterone on PND-8 and PND-9, levels of AR mRNA were also intermediate between those found in intact males and intact females. The exact time course for transcriptional regulation of AR mRNA in the developing rat brain is unknown. However, others have shown significant regulation of AR mRNA within hours of hormone treatment, so that 2 days of hormone withdrawal or replacement are probably sufficient to achieve new steady state levels of message. Moreover, sexually dimorphic neuronal loss has been documented to peak in hypothalamic cell groups during the first postnatal week. Thus, it is likely that changes in the number of AR mRNA-expressing cells as well as the amount of AR mRNA expression per cell are responsible for the development of male-typical AR mRNA content.

Sexually dimorphic ontogeny of GABAergic influences on periventricular somatostatin neurons.

The biosynthesis and secretion of somatostatin (SRIH) within the hypothalamic periventricular-median eminence (PeN-ME) pathway follows a sexually differentiated developmental pattern beginning in the early neonatal period. It is generally accepted that testosterone plays a role in these processes, but the mechanisms underlying the age and sex differences are poorly understood. The present study sought to investigate the hypothesis that gamma-aminobutyric acid (GABA) may play a role in determining sex differences in SRIH neuronal activity. Using an in vitro hypothalamic preparation where more than 97% of the immunoreactive SRIH is contained within the PeN-ME pathway, peptide release in response to the GABA(A) receptor antagonist, bicuculline, was followed through development. In the male a stimulatory response, indicative of an inhibitory GABAergic tone on SRIH secretion, was observed as early as postnatal day (P) 5. This persisted throughout juvenile development (P10, P17) and was present also in the adult male (P75), but in the peripubertal period the response to bicuculline was first lost (P25) and then reversed to an inhibition (P40), suggesting a transient switch to an apparent stimulatory GABAergic tone on SRIH release. By contrast, in the female, no bicuculline responsiveness was seen until P25 when it caused a decrease in SRIH release which persisted into adulthood. Using in situ hybridization studies we found no evidence to support the view that these age- and sex-dependent differences were due to changes in the expression of GABA(A) receptor alpha-subunits (alpha(1) and alpha(2)) which are colocalised in the PeN SRIH neurons. Following adult gonadectomy, the bicuculline response was abolished in the male, whereas, in the female it was reversed and identical in magnitude to the response in the intact male. These results demonstrate marked sex differences in GABA(A)-receptor-mediated influences on SRIH release which develop soon after birth and, in the adult, depend on gonadal factors. In the male these factors activate a primarily inhibitory influence, whereas in the female they facilitate an apparently stimulatory tone of GABA on SRIH secretion via the GABA(A) receptor. Our findings thus support the view that GABAergic transmission may play a key role in generating sex differences in the mode of SRIH secretion from the hypothalamus which has been shown to be a major factor in determining the sexually dimorphic patterns of growth hormone secretion.

Ontogeny of region-specific sex differences in androgen receptor messenger ribonucleic acid expression in the rat forebrain.

Testosterone and its metabolites are the principal gonadal hormones responsible for sexual differentiation of the brain. However, the relative roles of the androgen receptor (AR) vs. the estrogen receptor in specific aspects of this process remain unclear due to the intracellular metabolism of testosterone to active androgenic and estrogenic compounds. In this study, we used an 35S-labeled riboprobe and in situ hybridization to analyze steady state, relative levels of AR messenger RNA (mRNA) expression in the developing bed nucleus of the stria terminalis, medial preoptic area, and lateral septum, as well as the ventromedial and arcuate nuclei of the hypothalamus. Each area was examined on embryonic day 20 and postnatal days 0, 4, 10, and 20 to produce a developmental profile of AR mRNA expression. AR mRNA hybridization was present on embryonic day 20 in all areas analyzed. In addition, AR mRNA expression increased throughout the perinatal period in all areas examined in both males and females. However, between postnatal days 4 and 10, sharp increases in AR mRNA expression in the principal portion of the bed nucleus of the stria terminalis and the medial preoptic area occurred in the male that were not paralleled in the female. Subsequently, males exhibited higher levels of AR mRNA than females in these areas by postnatal day 10. There was no sex difference in AR mRNA content in the lateral septum, ventromedial nucleus, or arcuate nucleus at any age. These results suggest that sex differences in AR mRNA expression during development may lead to an early sex difference in sensitivity to the potential masculinizing effects of androgen.

Hormonal regulation of androgen receptor messenger RNA in the medial preoptic area of the male rat.

In the adult male rat, androgen and estrogen synergize in the regulation of male reproductive behaviors. To explore some of the molecular mechanisms underlying this synergism we examined the distribution and hormonal regulation of androgen receptor (AR) and estrogen receptor (ER) mRNAs in the medial preoptic area (MPOA) and bed nucleus of the stria terminalis (BST) of the adult male rat. Using in situ hybridization, AR and ER mRNAs were found to be distributed in overlapping but unique patterns. The highest density of AR mRNA was found in the central part of the medial preoptic n. and the principal n. of the BST. Gonadectomy (GDX) of adult male rats caused an increase in hybridization density in both brain areas after 4 days followed by a decrease after 2 months. In contrast, ER mRNA was increased following GDX and remained high regardless of length of time. Treatment of adult GDX'd males with dihydrotestosterone (DHT) reversed the effects of GDX on AR mRNA at both the short and long-term castrate but had no effect on ER mRNA in both the MPOA and BST. Estrogen treatment increased AR mRNA in the long-term castrate only and decreased ER mRNA in both long- and short-term castrates. Immunocytochemical detection of AR revealed a similar distribution to AR mRNA; however, AR immunoreactivity was reduced in the MPOA and BST after both short- and long-term GDX. In vitro [3H]DHT binding in cytosols of the preoptic area showed appreciable binding but there was no effect of length of time following GDX. These data show that the pattern of regulation of AR mRNA is unique to this receptor type and does not follow the pattern of regulation of the ER mRNA. Furthermore, although the distribution of AR mRNA and AR protein coincide within the MPOA, changes in mRNA levels as a result of castration or hormone treatment do not result in corresponding changes in binding. This mismatch between mRNA and binding suggests a complex regulation of AR beyond simply changes in transcription.

Developmental profile and regulation of estrogen receptor (ER) mRNA expression in the preoptic area of prenatal rats.

Estrogen, derived from circulating testosterone, masculinizes the developing preoptic area. Expression of estrogen receptors (ERs) within the preoptic area is one requirement for a possible direct action of estrogen in the process of sexual differentiation of this brain region. Using a 35S-labeled riboprobe and in situ hybridization to detect ER mRNA on both film and emulsion-coated slides, we were able to detect ER mRNA within the rat preoptic area by embryonic day 18 (ED 18), coincident with the reported onset of the critical period for testosterone-dependent masculinization of this region. ER mRNA increased significantly between ED 18 and 19 in both sexes, and continued to increase through postnatal day 0 (PND 0 = day of birth) in females, but not males. ER mRNA levels were not significantly greater in females than in males until PND 0. The lack of a sex difference in ER mRNA prenatally, however, appears to be due to an effect of intrauterine neighbors. ER mRNA levels in ED 20 embryos were relatively high in females with female-only neighbors, whereas ER mRNA levels were relatively low, and comparable to males, when the in utero neighbors included one or more males. Treatment of pregnant dams with diethylstilbestrol or with tamoxifen did not significantly alter ER mRNA levels in the preoptic area of the embryos. Although these results suggest that ER mRNA expression is subject to hormonal regulation prenatally, the relevant hormone was not identified.

Region-specific effects of ovarian hormones on estrogen receptor immunoreactivity.

The cell groups in which nuclear estrogen receptor (ER) expressing neurons are found have unique, often coordinated, functions. Regulation of ER content may be one mechanism through which feedback responses can be adjusted to match the function of a specific brain region and physiological circumstance. In these immunocytochemical experiments, estrogen decreased staining intensity for ER in the ventrolateral hypothalamus and bed nucleus of the stria terminalis, but not in the periventricular preoptic area. ER staining intensity was further decreased by progesterone, following estrogen, but not in all brain regions. These results suggest that ER is regulated by estrogen in a region-specific manner. Furthermore, inhibition of responses to estrogen by progesterone may involve progesterone-induced down-regulation of ERs.

Estrogen receptor immunoreactivity in ferret brain is regulated by estradiol in a region-specific manner.

The effects of estrogen on estrogen receptor (ER) immunoreactivity in the male ferret brain were examined. Estrogen treatment reduced the mean number of ER-immunopositive (ER+) cells/unit area in periventricular preoptic area but increased the mean number of ER+ cells/unit area in the medial division of the ventromedial hypothalamic nucleus, while having no effect on the number of ER+ cells/unit area in the lateral VMH and arcuate nucleus. Thus, estrogen regulates brain ER immunoreactivity in male ferrets and the direction and magnitude of this regulation are brain region-specific.

Estrogen receptor mRNA levels in the preoptic area of neonatal rats are responsive to hormone manipulation.

Testosterone, after conversion to estrogen, masculinizes the developing preoptic area (POA) of rats, via binding to intracellular estrogen receptors located within the POA. Our previous studies have shown what seems to be a paradox, in that the levels of estrogen receptor mRNA are lower in males than in females. In the present study, we examined the effects of hormone manipulations on estrogen receptor (ER) mRNA levels in the preoptic area of neonatal male and female rats to test the hypothesis that gonadal steroid hormones regulate ER mRNA during the perinatal period. The relative amount of steady state ER mRNA was assessed in the preoptic area of postnatal day 4 animals using in situ hybridization and film autoradiography. Hybridization density was approximately 2-fold higher in females compared with hybridization density in males. Depletion of testosterone by bilateral removal of the testes on the day of birth increased the level of ER mRNA in males to that observed in females. Treatment of females with the synthetic estrogen, diethylstilbestrol (1 microgram per day, in pellet form), reduced ER mRNA levels to a level comparable to that in intact males. The non-aromatizable androgen, dihydrotestosterone (50 micrograms per day, in pellet form), had no effect on ER mRNA in females. These results suggest that estrogen, derived from the local aromatization of circulating testosterone, down-regulates ER mRNA in the neonatal male preoptic area. Down-regulation of ER mRNA may be an important estrogen-regulated event in the process of sexual differentiation of the preoptic area.

Developmental profile of estrogen receptor mRNA in the preoptic area of male and female neonatal rats.

Exposure to estrogen or estrogenic metabolites of testosterone during the early postnatal period has permanent effects on rodent brain development. Differential sensitivity to estrogen, as reflected by transcription of the estrogen receptor gene, might determine the period of maximal sensitivity to the masculinizing effects of estrogen. We used an 35S-labeled riboprobe and in situ hybridization to chart the development of estrogen receptor (ER) mRNA expression in the rat preoptic area, a brain region for which sexual dimorphisms and the effects of estrogen on development are particularly well documented. Neonatal male and female rats were sacrificed by perfusion fixation on postnatal days 0, 2, 4, 7 or 10 (PND; day of birth is PND 0). Many ER mRNA-containing cells were detected in the periventricular preoptic area and medical preoptic nucleus and the distribution of ER-synthesizing cells was similar in both sexes. Analysis of film autoradiograms showed that the relative steady state level of ER mRNA was significantly higher in females than in males at all ages except PND 0 and 10. The temporal profile of ER mRNA expression was different in males and females. ER mRNA did not change with age in males, whereas in females, ER mRNA was significantly higher on PND 2 compared with PND 0 and 10. These results demonstrate that the pattern of ER mRNA expression is quantitatively and qualitatively different between the sexes during the neonatal period. The pattern of ER mRNA expression contrasts markedly with previous reports of estrogen binding based on biochemical and autoradiographic steroid binding assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen receptor immunoreactivity in specific brain areas of the prairie vole (Microtus ochrogaster) is altered by sexual receptivity and genetic sex.

The prairie vole is a small rodent with an unusual reproductive strategy. A sexually naive female vole requires male contact to initiate the maturation of her reproductive functions. Contact with an unfamiliar adult male vole increases blood estrogen levels, reproductive tissue weights, and brain nuclear estrogen receptor binding levels of female voles. What is not known is: 1) What is the precise distribution of estrogen receptor containing neurons in the prairie vole brain? 2) Does male induced sexual receptivity alter the distribution or number of estrogen receptors in specific brain areas of the female vole? 3) Do male and female voles differ in the distribution or number of estrogen receptor containing neurons? We compared sexually receptive-male-exposed females, sexually naive females, and sexually naive males, for the presence of estrogen receptor immunoreactive (ER-IR) neurons in specific cell groups of the brain. The number of ER-IR neurons per cell group was counted and the relative amount of immunoreactivity per neuron was measured by densitometry. The neuroanatomical distribution of estrogen receptor containing neurons in the vole was similar to the distribution of estrogen receptors in most rodents. The mean number of ER-IR neurons did not differ between naive and male-exposed females. The induction of sexual receptivity however significantly decreased the concentration of estrogen receptor immunoreactivity per neuron in the medial preoptic nucleus, the medial preoptic area, the encapsulated bed nucleus of the stria terminalis, and the ventromedial nucleus of the hypothalamus. Compared with naive males, the mean number of ER-IR neurons was up to four fold greater in naive females in the medial preoptic nucleus, anteroventral periventricular preoptic nucleus, the encapsulated bed nucleus of the stria terminalis, the medial amygdala, and the ventromedial nucleus of the hypothalamus. Additionally the amount of estrogen receptor immunoreactivity per neuron was considerably greater in the medial preoptic nucleus, the medial preoptic area, the encapsulated bed nucleus of the stria terminalis, and the ventromedial nucleus of the hypothalamus of naive females. If the amount of estrogen receptor per cell is a determinant of a tissue's responsiveness to estrogen, reduced estrogen receptor immunoreactivity in males, and in females exposed to males suggests that they may be less responsive to estrogen than naive females. We propose that this reduced estrogen receptor immunoreactivity in males is a result of reduced estrogen receptor protein levels. Currently, we cannot definitively prove our working hypothesis that decreased estrogen receptor immunoreactivity in females exposed to males is due to reduced receptor levels, and not due to ligand altered epitope availability.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of estrogen receptor-immunoreactive cells in the forebrain of the female guinea pig.

We mapped the distribution of estrogen receptor-containing cells in the forebrain of the adult female guinea pig. Cellular estrogen receptor content was detected using monoclonal antibody H222, directed against the estrogen receptor, and the avidin-biotin method with nickel-intensified diaminobenzidine as the chromagen. A complete set of deletion, titration, and adsorption controls established the specificity of the staining. The most dense collections of estrogen receptor-immunoreactive cells were found in medial preoptic, medial hypothalamic, and limbic nuclei (amygdala, bed nucleus of the stria terminalis, lateral septum). Numerous estrogen receptor-immunoreactive cells were also found in additional, specific subregions of the remainder of the preoptic area, hypothalamus, and limbic system, and also in the midbrain (central gray). Elsewhere, estrogen receptor-immunoreactive cells were present in smaller numbers or were absent. This map confirms and extends previous maps based on estrogen binding. The majority of estrogen receptor-immunoreactive cells are found in areas known to be involved in some aspect of reproduction. In addition, many estrogen receptor-immunoreactive cells are found in areas not typically considered to have a primary role in reproductive behavior or neuroendocrine function.

Ultrastructural characteristics of estrogen receptor-containing neurons of the ventrolateral nucleus of the Guinea-pig hypothalamus.

Abstract The ventrolateral nucleus of the hypothalamus (VL) of the guinea-pig is a key cell group in the neural circuitry underlying the estrogen-dependent lordosis reflex. The extent to which neurons in the VL are responsive to estrogen or to synaptic inputs depends in part on the presence of specific estrogen and neurotransmitter receptors within the target cells. It also depends on the number, type and location of synaptic inputs. In addition, both sensitivity to circulating hormones and transmitter responsiveness show estrogen-inducible alterations in the VL. To understand more about the cell types that are directly modulated by estrogens via the nuclear steroid binding protein and the synaptic connectivity of these neurons, we have carried out an ultrastructural study of estrogen receptor-containing cells in the VL of the female guinea-pig. Estrogen receptor was localized for both light and electron microscopy using a specific monoclonal antibody, H-222, directed against the human estrogen receptor. Numerous immunoreactive neurons were found in the VL. These cells had simple, relatively smooth dendritic processes that were generally unbranched. Reaction product was most intense in the nucleus; lighter deposits were seen in some but not all somata and proximal dendrites. No cell was observed with only cytoplasmic staining. At the ultrastructural level, this distribution of reaction product within cells was confirmed. Gold deposits were associated with euchromatin and excluded from the nucleolus, nucleolar-associated heterochromatin and Barr body. In the cytoplasm, the small aggregates of gold particles were randomly distributed. Two types of cytologically distinct immunoreactive neurons were characterized. The most numerous category was of large cells with extensive rough endoplasmic reticulum, frequently organized as whorls or ribbons, several stacks of Golgi cisterna, numerous mitochondria and multivesicular bodies. A smaller population, representing approximately 5% of the total, was of much smaller cells which had only a thin rim of cytoplasm around the nucleus, scattered elements of the rough endoplasmic reticulum and a single small Golgi saccule. Based on size, we suggest that the larger neurons are projection neurons and that the smaller ones form local circuits. The larger cells received a dense axo-somatic and axo-dendritic innervation. Most of the presynaptic terminals contained small, clear round vesicles; synaptic densities on either pre- or postsyn- aptic side were absent though a well defined synaptic cleft was evident. Very few synapses were found on the small cells even when serial sections were examined. It is clear that the larger, estrogen receptor-containing neurons are in a position to integrate both hormonal and neuronal signals and to transmit this information to other regions of the central nervous system involved in the outflow of reproductive behaviors.