Neurons Express Estrogen Receptor Research Articles

Short-term depletion of plasma estrogen affects hypothalamic kisspeptin-neurokinin B-dynorphin A neurons, gonadotrophs, and pulsatile luteinizing hormone secretion in female rats

Kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the arcuate nucleus (ARC) regulate pulsatile luteinizing hormone (LH) secretion. These neurons express estrogen receptors and are negatively regulated by estrogen. This study aimed to determine whether estrogen supplementation after short-term ovariectomy-induced estrogen depletion has different effects on KNDy neurons depending on the timing of the supplementation. To decrease endogenous estradiol (E2) for a short time, adult female rats received a tube filled with E2 one week after ovariectomy and utilized it one week later (O1w + E). From the results of immunohistochemistry, the response to E2 was attenuated in KNDy neurons of O1w + E rats. Enlarged LH-secreting cells in the anterior pituitary were found in O1w + E rats; however, such enlarged LH cells were not found in ones without previous short-term E2 depletion. From the analysis of LH pulses, plasma LH levels were increased in O1w + E rats relative to ones without previous short-term E2 depletion. These results suggested that once endogenous sex steroids were depleted, the response to E2 in hypothalamic KNDy neurons did not fully recover in one week. Thus, short-term sex steroid depletion due to gonadectomy could alter the response to the sex steroids in KNDy neurons even though the period without sex steroids is only one week, and the alteration is likely to affect plasma hormone levels.

DNA Methylation and Demethylation Underlie the Sex Difference in Estrogen Receptor Alpha in the Arcuate Nucleus

Introduction: Neurons expressing estrogen receptor (ER) ɑ in the arcuate (ARC) and ventromedial (VMH) nuclei of the hypothalamus sex-specifically control energy homeostasis, sexual behavior, and bone density. Females have more ERɑ neurons in the VMH and ARC than males, and the sex difference in the VMH is eliminated by neonatal treatment with testosterone or a DNA methylation inhibitor. Objective: Here, we tested the roles of testosterone and DNA methylation/demethylation in development of ERɑ in the ARC. Methods: ERɑ was examined at birth and weaning in mice that received vehicle or testosterone subcutaneously, and vehicle or DNA methyltransferase inhibitor intracerebroventricularly, as neonates. To examine effects of DNA demethylation on the ERɑ cell number in the ARC, mice were treated neonatally with small interfering RNAs against ten-eleven translocase enzymes. The methylation status of the ERɑ gene (Esr1) was determined in the ARC and VMH using pyrosequencing of bisulfite-converted DNA. Results: A sex difference in ERɑ in the ARC, favoring females, developed between birth and weaning and was due to programming effects of testosterone. Neonatal inhibition of DNA methylation decreased ERɑ in the ARC of females, and an inhibition of <underline>de</underline>methylation increased ERɑ in the ARC of males. The promoter region of Esr1 exhibited a small sex difference in percent of total methylation in the ARC (females > males) that was opposite to that in the VMH (males > females). Conclusion: DNA methylation and demethylation regulate ERɑ cell number in the ARC, and methylation correlates with activation of Esr1 in this region.

ERα Signaling in GHRH/Kiss1 Dual-Phenotype Neurons Plays Sex-Specific Roles in Growth and Puberty.

Gonadal steroids modulate growth hormone (GH) secretion and the pubertal growth spurt via undefined central pathways. GH-releasing hormone (GHRH) neurons express estrogen receptor α (ERα) and androgen receptor (AR), suggesting changing levels of gonadal steroids during puberty directly modulate the somatotropic axis. We generated mice with deletion of ERα in GHRH cells (GHRHΔERα), which displayed reduced body length in both sexes. Timing of puberty onset was similar in both groups, but puberty completion was delayed in GHRHΔERα females. Lack of AR in GHRH cells (GHRHΔAR mice) induced no changes in body length, but puberty completion was also delayed in females. Using a mouse model with two reporter genes, we observed that, while GHRHtdTom neurons minimally colocalize with Kiss1hrGFP in prepubertal mice, ∼30% of GHRH neurons coexpressed both reporter genes in adult females, but not in males. Developmental analysis of Ghrh and Kiss1 expression suggested that a subpopulation of ERα neurons in the arcuate nucleus of female mice undergoes a shift in phenotype, from GHRH to Kiss1, during pubertal transition. Our findings demonstrate that direct actions of gonadal steroids in GHRH neurons modulate growth and puberty and indicate that GHRH/Kiss1 dual-phenotype neurons play a sex-specific role in the crosstalk between the somatotropic and gonadotropic axes during pubertal transition.SIGNIFICANCE STATEMENT Late maturing adolescents usually show delayed growth and bone age. At puberty, gonadal steroids have stimulatory effects on the activation of growth and reproductive axes, but the existence of gonadal steroid-sensitive neuronal crosstalk remains undefined. Moreover, the neural basis for the sex differences observed in the clinical arena is unknown. Lack of ERα in GHRH neurons disrupts growth in both sexes and causes pubertal delay in females. Deletion of androgen receptor in GHRH neurons only delayed female puberty. In adult females, not males, a subset of GHRH neurons shift phenotype to start producing Kiss1. Thus, direct estrogen action in GHRH/Kiss1 dual-phenotype neurons modulates growth and puberty and may orchestrate the sex differences in endocrine function observed during pubertal transition.

Neurochemical Characterization of Neurons Expressing Estrogen Receptor β in the Hypothalamic Nuclei of Rats Using in Situ Hybridization and Immunofluorescence.

Estrogens play an essential role in multiple physiological functions in the brain, including reproductive neuroendocrine, learning and memory, and anxiety-related behaviors. To determine these estrogen functions, many studies have tried to characterize neurons expressing estrogen receptors known as ERα and ERβ. However, the characteristics of ERβ-expressing neurons in the rat brain still remain poorly understood compared to that of ERα-expressing neurons. The main aim of this study is to determine the neurochemical characteristics of ERβ-expressing neurons in the rat hypothalamus using RNAscope in situ hybridization (ISH) combined with immunofluorescence. Strong Esr2 signals were observed especially in the anteroventral periventricular nucleus (AVPV), bed nucleus of stria terminalis, hypothalamic paraventricular nucleus (PVN), supraoptic nucleus, and medial amygdala, as previously reported. RNAscope ISH with immunofluorescence revealed that more than half of kisspeptin neurons in female AVPV expressed Esr2, whereas few kisspeptin neurons were found to co-express Esr2 in the arcuate nucleus. In the PVN, we observed a high ratio of Esr2 co-expression in arginine-vasopressin neurons and a low ratio in oxytocin and corticotropin-releasing factor neurons. The detailed neurochemical characteristics of ERβ-expressing neurons identified in the current study can be very essential to understand the estrogen signaling via ERβ.

Neonatal Inhibition of DNA Methylation Disrupts Testosterone-Dependent Masculinization of Neurochemical Phenotype.

Many neural sex differences are differences in the number of neurons of a particular phenotype. For example, male rodents have more calbindin-expressing neurons in the medial preoptic area (mPOA) and bed nucleus of the stria terminalis (BNST), and females have more neurons expressing estrogen receptor alpha (ERα) and kisspeptin in the ventromedial nucleus of the hypothalamus (VMH) and the anteroventral periventricular nucleus (AVPV), respectively. These sex differences depend on neonatal exposure to testosterone, but the underlying molecular mechanisms are unknown. DNA methylation is important for cell phenotype differentiation throughout the developing organism. We hypothesized that testosterone causes sex differences in neurochemical phenotype via changes in DNA methylation, and tested this by inhibiting DNA methylation neonatally in male and female mice, and in females given a masculinizing dose of testosterone. Neonatal testosterone treatment masculinized calbindin, ERα and kisspeptin cell number of females at weaning. Inhibiting DNA methylation with zebularine increased calbindin cell number only in control females, thus eliminating sex differences in calbindin in the mPOA and BNST. Zebularine also reduced the sex difference in ERα cell number in the VMH, in this case by increasing ERα neuron number in males and testosterone-treated females. In contrast, the neonatal inhibition of DNA methylation had no effect on kisspeptin cell number. We conclude that testosterone normally increases the number of calbindin cells and reduces ERα cells in males through orchestrated changes in DNA methylation, contributing to, or causing, the sex differences in both cell types.

Neurons expressing estrogen receptor α differentially innervate the periaqueductal gray matter of female rats

The periaqueductal gray matter (PAG) is a brainstem site involved in distinct autonomic and behavioral responses. Among them, the motor control of female sexual behavior, including lordosis, is well described. Lordosis reflex is highly dependent on increasing levels of estradiol that occur in the afternoon of the proestrus day in normally cycling females. This effect is thought to be mediated primarily via actions in the ventromedial nucleus of the hypothalamus (VMH). By binding to estrogen receptor α (ERα), estradiol changes the activity of VMH neurons that project to the PAG. Evidence also exists for the coordination of PAG outputs by estradiol-responsive neurons outside the VMH. However, a comprehensive analysis of these circuitries is not available. Using stereotaxic injection of the retrograde tracer Fluorogold in distinct columns of the PAG we performed a systematic mapping of neurons innervating the PAG and those coexpressing ERα immunoreactivity. We found that the forebrain projections to PAG columns are largely segregated and that most of the ERα expressing neurons preferentially target the lateral and the ventrolateral columns. Dual labeled neurons were mostly found in the intermediate subdivision of the lateral septal nucleus, the posterior aspect of the medial bed nucleus of the stria terminalis, the medial preoptic nucleus, the striohypothalamic nucleus and the ventrolateral VMH. Few dual labeled neurons were also observed in the arcuate nucleus, in the posterodorsal subdivision of the medial nucleus of the amygdala and in the ventral premammillary nucleus. Our findings indicate that ERα modulates sexual behavior in female rats via an integrated neural network that differentially innervate the columns of the PAG.

Voluntary urination control by brainstem neurons that relax the urethral sphincter.

Voluntary urination ensures that waste is eliminated when safe and socially appropriate, even without a pressing urge. Uncontrolled urination, or incontinence, is a common problem with few treatment options. Normal urine release requires a small region in the brainstem known as Barrington’s nucleus (Bar), but specific neurons that relax the urethral sphincter and enable urine flow are unknown. Here we identify a small subset of novel Bar neurons that control the urethral sphincter in mice. These excitatory neurons express estrogen receptor 1 (BarESR1), project to sphincter-relaxing interneurons in the spinal cord and are active during natural urination. Optogenetic stimulation of BarESR1 neurons rapidly initiates sphincter bursting and efficient voiding in anesthetized and behaving animals. Conversely, optogenetic and chemogenetic inhibition reveals their necessity in motivated urination behavior. The identification of these cells provides an expanded model for the control of urination and its dysfunction.

Medial preoptic area in mice is capable of mediating sexually dimorphic behaviors regardless of gender

The medial preoptic area (mPOA) differs between males and females in nearly all species examined to date, including humans. Here, using fiber photometry recordings of Ca2+ transients in freely behaving mice, we show ramping activities in the mPOA that precede and correlate with sexually dimorphic display of male-typical mounting and female-typical pup retrieval. Strikingly, optogenetic stimulation of the mPOA elicits similar display of mounting and pup retrieval in both males and females. Furthermore, by means of recording, ablation, optogenetic activation, and inhibition, we show mPOA neurons expressing estrogen receptor alpha (Esr1) are essential for the sexually biased display of these behaviors. Together, these results underscore the shared layout of the brain that can mediate sex-specific behaviors in both male and female mice and provide an important functional frame to decode neural mechanisms governing sexually dimorphic behaviors in the future.

Morphological Analysis of the Axonal Projections of EGFP-Labeled Esr1-Expressing Neurons in Transgenic Female Medaka.

Some hypothalamic neurons expressing estrogen receptor α (Esr1) are thought to transmit a gonadal estrogen feedback signal to gonadotropin-releasing hormone 1 (GnRH1) neurons, which is the final common pathway for feedback regulation of reproductive functions. Moreover, estrogen-sensitive neurons are suggested to control sexual behaviors in coordination with reproduction. In mammals, hypothalamic estrogen-sensitive neurons release the peptide kisspeptin and regulate GnRH1 neurons. However, a growing body of evidence in nonmammalian species casts doubt on the regulation of GnRH1 neurons by kisspeptin neurons. As a step toward understanding how estrogen regulates neuronal circuits for reproduction and sex behavior in vertebrates in general, we generated a transgenic (Tg) medaka that expresses enhanced green fluorescent protein (EGFP) specifically in esr1-expressing neurons (esr1 neurons) and analyzed their axonal projections. We found that esr1 neurons in the preoptic area (POA) project to the gnrh1 neurons. We also demonstrated by transcriptome and histological analyses that these esr1 neurons are glutamatergic or γ-aminobutyric acidergic (GABAergic) but not kisspeptinergic. We therefore suggest that glutamatergic and GABAergic esr1 neurons in the POA regulate gnrh1 neurons. This hypothesis is consistent with previous studies in mice that found that glutamatergic and GABAergic transmission is critical for estrogen-dependent changes in GnRH1 neuron firing. Thus, we propose that this neuronal circuit may provide an evolutionarily conserved mechanism for regulation of reproduction. In addition, we showed that telencephalic esr1 neurons project to medulla, which may control sexual behavior. Moreover, we found that some POA-esr1 neurons coexpress progesterone receptors. These neurons may form the neuronal circuits that regulate reproduction and sex behavior in response to the serum estrogen/progesterone.

Classical and membrane-initiated estrogen signaling in an in vitro model of anterior hypothalamic kisspeptin neurons.

The neuropeptide kisspeptin is essential for sexual maturation and reproductive function. In particular, kisspeptin-expressing neurons in the anterior rostral periventricular area of the third ventricle are generally recognized as mediators of estrogen positive feedback for the surge release of LH, which stimulates ovulation. Estradiol induces kisspeptin expression in the neurons of the rostral periventricular area of the third ventricle but suppresses kisspeptin expression in neurons of the arcuate nucleus that regulate estrogen-negative feedback. To focus on the intracellular signaling and response to estradiol underlying positive feedback, we used mHypoA51 cells, an immortalized line of kisspeptin neurons derived from adult female mouse hypothalamus. mHypoA51 neurons express estrogen receptor (ER)-α, classical progesterone receptor (PR), and kisspeptin, all key elements of estrogen-positive feedback. As with kisspeptin neurons in vivo, 17β-estradiol (E2) induced kisspeptin and PR in mHypoA51s. The ERα agonist, 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, produced similar increases in expression, indicating that these events were mediated by ERα. However, E2-induced PR up-regulation required an intracellular ER, whereas kisspeptin expression was stimulated through a membrane ER activated by E2 coupled to BSA. These data suggest that anterior hypothalamic kisspeptin neurons integrate both membrane-initiated and classical nuclear estrogen signaling to up-regulate kisspeptin and PR, which are essential for the LH surge.

Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor α in kisspeptin neurons.

Hypothalamic kisspeptin (Kiss1) neurons express estrogen receptor α (ERα) and exert control over GnRH/LH secretion in female rodents. It has been proposed that estradiol (E2) activation of ERα in kisspeptin neurons in the arcuate nucleus (ARC) suppresses GnRH/LH secretion (negative feedback), whereas E2 activation of ERα in kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) mediates the release of preovulatory GnRH/LH surges (positive feedback). To test these hypotheses, we generated mice bearing kisspeptin cell-specific deletion of ERα (KERαKO) and treated them with E2 regimens that evoke either negative or positive feedback actions on GnRH/LH secretion. Using negative feedback regimens, as expected, E2 effectively suppressed LH levels in ovariectomized (OVX) wild-type (WT) mice to the levels seen in ovary-intact mice. Surprisingly, however, despite the fact that E2 regulation of Kiss1 mRNA expression was abrogated in both the ARC and AVPV of KERαKO mice, E2 also effectively decreased LH levels in OVX KERαKO mice to the levels seen in ovary-intact mice. Conversely, using a positive feedback regimen, E2 stimulated LH surges in WT mice, but had no effect in KERαKO mice. These experiments clearly demonstrate that ERα in kisspeptin neurons is required for the positive, but not negative feedback actions of E2 on GnRH/LH secretion in adult female mice. It remains to be determined whether the failure of KERαKO mice to exhibit GnRH/LH surges reflects the role of ERα in the development of kisspeptin neurons, in the active signaling processes leading to the release of GnRH/LH surges, or both.

Evidence that histaminergic neurons are devoid of estrogen receptor alpha in the ewe diencephalon during the breeding season

In sheep as in rat, it has been highly suggested that neuronal histamine (HA) participates to the estradiol (E2)-induced GnRH and LH surges, through H1 receptor. With the aim of determining if E2 could act directly on HA neurons, we examined here whether HA neurons express estrogen receptor alpha (ERα) in the ewe diencephalon during the breeding season. We first produced a specific polyclonal antibody directed against recombinant ovine histidine decarboxylase (oHDC), the HA synthesizing enzyme. Using both this anti-oHDC antibody and an anti-ERα monoclonal antibody in double label immunohistochemistry, we showed that HA neurons do not express ERα in diencephalon of ewes with different hormonal status. This result diverges from those obtained in rat, in which around three quarters of HA neurons express ERα in their nucleus. This discrepancy between these two mammal species may reflect difference in their neuronal network.

Modification of nitric oxide synthase immunoreactivity in female mouse limbic system during the estrous cycle

Nitric oxide (NO) is a gaseous neuronal messenger, synthesized by the enzyme nitric oxide synthase (NOS), and implicated in the regulation of several physiological and behavioral functions. NO-producing neurons have been localized in numerous regions of the mammalian and non-mammalian central nervous system. In rodents, NOS-immunoreactive neurons and fibers were described in hypothalamic and limbic nuclei belonging to neural circuits implicated in the control of reproductive behavior. Fluctuating levels of estradiol and progesterone during the estrous cycle may induce structural changes in several brain nuclei, where some neurons express estrogen receptors. Various reports indicate that the expression of nNOS is influenced by estrogens in the female. Therefore, to clarify if the nitrergic system is a target for gonadal hormones in physiological conditions, we have investigated the effects of estrous cycle in the expression of nNOS immunoreactivity in different nuclei of the limbic system on two-month-old intact female mice. Changes were observed in the medial preoptic area (significantly higher number in estrus) and in the arcuate nucleus (significantly higher number in proestrus). In the ventrolateral part of the ventromedial nucleus and in the bed nucleus of the stria terminalis no significant changes have been observed. In hippocampus the number of nNOS positive neurons fluctuates during the estrous cycle, reaching its peak during proestrus and metaestrus, and these variations were statistically significant. These results suggest that, in mice, the expression of nNOS in some limbic regions, involved in the control of reproduction and characterized by a large number of estrogen receptors, is under the control of gonadal hormones and may vary according to the short-term variations of hormonal levels that take place during the estrous cycle.? This study has been supported by grants from MURST and University of Torino (SG and GCP).

Estrogen receptor α is involved in the estrogenic regulation of arginine vasopressin immunoreactivity in the supraoptic and paraventricular nuclei of ovariectomized rats

The ovarian hormone estradiol regulates the expression of arginine vasopressin gene and the release of arginine vasopressin by magnocellular hypothalamic neurons. Magnocellular neurons express estrogen receptor β and are contacted by afferent neurons that express estrogen receptor α. In this study we have assessed the effect of selective ligands for estrogen receptors to determine the subtype of estrogen receptor involved in the regulation of arginine vasopressin immunoreactivity in the supraoptic and paraventricular nuclei of ovariectomized rats. The volume fraction occupied by arginine vasopressin immunoreactive material was significantly increased in both nuclei in the animals treated with estradiol compared to the animals injected with vehicle. A similar result was obtained with an estrogen receptor α selective agonist. In contrast, the administration of an estrogen receptor β selective agonist did not significantly affect arginine vasopressin immunoreactivity. This finding suggests that estradiol may regulate arginine vasopressin levels on the supraoptic and paraventricular nuclei by acting on afferent neurons expressing estrogen receptor α.

Effects of Estradiol on Cerebrospinal Fluid Levels of Agouti-Related Protein in Ovariectomized Rhesus Monkeys

Hypothalamic proopiomelanocortin (POMC)-derived MSH peptides and the melanocortin receptor antagonist, agouti-related protein (AgRP), interact to regulate energy balance. Both POMC and AgRP neurons express estrogen receptors, but little is known about estrogen regulation of the melanocortin system in the primate. We have therefore examined the effects of physiological doses of estradiol (E2) on POMC and AgRP in lumbar cerebrospinal fluid (CSF) of ovariectomized monkeys. POMC prohormone was measured by ELISA. AgRP was measured by RIA (sensitive for the more biologically active C-terminal AgRP(83-132) but also detects full-length AgRP) and by ELISA (measures primarily full length AgRP). In the first experiment, 14 animals were studied before and after 3 wk of E2. CSF POMC did not change, but AgRP(RIA) decreased from 7.9 +/- 1.2 to 4.7 +/- 1.2 fmol/ml after E2 (P = 0.03) and the POMC/AgRP(RIA) ratio increased from 4.2 +/- 0.89 to 6.8 +/- 1.04 (P = 0.04). AgRP(ELISA) did not change, but the ratio of AgRP(RIA) compared with AgRP(ELISA) was reduced after E2 (P = 0.02). In the second experiment, 11 animals were studied after 6 wk of E2, and similar changes were noted. The degree of AgRP(RIA) suppression with E2 was inversely related to body mass index (r = 0.569; P = 0.03). These results show for the first time that E2 suppresses AgRP(C-terminal) in CSF, increases the POMC to AgRP ratio, and may decrease AgRP processing, thus leading to increased melanocortin signaling. Furthermore, obesity was associated with resistance to the suppressive effects of E2 on AgRP, analogous to what is seen with obesity and leptin resistance.

Rapid changes on nitrinergic system in female mouse hippocampus during the ovarian cycle

Fluctuating levels of estradiol and progesterone during the estrous cycle may induce structural changes in several brain nuclei including the hippocampus, where some neurons express estrogen receptors. Nitric oxide plays a wide range of functions in the nervous system generally by acting as a neurotransmitter-like molecule. It has been demonstrated that long-term treatments with estradiol in ovariectomized females and with testosterone in castrated males induce neuronal nitric oxide synthase (nNOS) expression in rat hypothalamus, whereas changes in nNOS immunoreactivity or in associated NADPH-diaphorase activity were observed both in hypothalamus and in amygdala during different phases of estrous cycle. Estradiol could induce nNOS expression in several brain regions in rodents. Therefore, to clarify if the hippocampal NO producing system is a target for gonadal hormones in physiological conditions, we have investigated the effects of estrous cycle in the expression of nNOS immunoreactivity on two-month-old intact female mice. Immunoreactive cells were observed in all hippocampal subregions: the higher number was detected in the pyramidal layer of CA1 region and in polymorph layer of dentate gyrus. The number of nNOS positive neurons fluctuates during the estrous cycle, reaching its peak during proestrus and metaestrus, and these variations were statistically significant in CA1, CA2 and CA3 regions. These results suggest that the nitrinergic system is a target for estrogen action in the hippocampus, and that this action may take place in physiological conditions according to the short-term variations of gonadal hormones during the estrous cycle.

Development of neurons expressing estrogen receptor α transiently in facial nucleus of prenatal and postnatal rat brains

The transient expression of estrogen receptor α (ERα) in the facial nucleus of rats during development was already reported. However, how and whether the receptor functions physiologically in the nucleus of developing rats are as yet unclear. In this study, we applied a retrograde tracer into one of the possible target muscles of the motoneurons in the nucleus, that is, the transverse auricular muscle (Mta), and examined whether ERα-immunopositive neurons take up the tracer. Because it is probable that neurogenesis, apoptosis, and maturation may be associated with the transient expression of ERα, we attempted to analyze the neurons expressing the receptor in the nucleus. We found that ERα-immunopositive neurons in the medial facial subnucleus innervate mostly the Mta. Quantitative analyses showed that the number of motoneurons projecting to the Mta remained the same throughout the ages examined, whereas that of ERα-immunopositive neurons decreased between postnatal days 6 and 11. Apoptosis and neurogenesis in the nucleus were not affected by the expression of ERα during development. ERα expression coincided with the maturation of neurons in the nucleus. Thus, it is possible that ERα expression in the facial nucleus during development plays important roles in the development of motoneurons and/or external pinna muscles.

Estrogen receptor-α mediates estradiol attenuation of ATP-induced Ca2+ signaling in mouse dorsal root ganglion neurons

A mechanism underlying gender-related differences in pain perception may be estrogen modulation of nociceptive signaling in the peripheral nervous system. In rat, dorsal root ganglion (DRG) neurons express estrogen receptors (ERs) and estrogen rapidly attenuates ATP-induced Ca2+ signaling. To determine which estrogen receptor mediates rapid actions of estrogen, we showed ERalpha and ERbeta expression in DRG neurons from wild-type (WT) female mice by RT-PCR. To study whether ERalpha or ERbeta mediates this response, we compared estradiol action mediating Ca2+ signaling in DRG neurons from WT, ERalpha knockout (ERalphaKO), and ERbetaKO mice in vitro. ATP, an algesic agent, induced [Ca2+]i transients in 48% of small DRG neurons from WT mice. 17beta-Estradiol (E2) inhibited ATP-induced intracellular Ca2+ concentration ([Ca2+]i) with an IC50 of 27 nM. The effect of E2 was rapid (5-min exposure) and stereo specific; 17alpha-estradiol had no effect. E2 action was blocked by the ER antagonist ICI 182,780 (1 microM) in WT mouse. Estradiol coupled to bovine serum albumin (E-6-BSA), which does not penetrate the plasma membrane, had the same effect as E2 did, suggesting that a membrane-associated ER mediated the response. In DRG neurons from ERbetaKO mice, E2 attenuated the ATP-induced [Ca2+]i flux as it did in WT mice, but in DRG neurons from ERalphaKO mice, E2 failed to inhibit the ATP-induced [Ca2+]i increase. These results show that mouse DRG neurons express ERs and the rapid attenuation of ATP-induced [Ca2+]i signaling is mediated by membrane-associated ERalpha.

Rapid actions of estradiol on cyclic amp response-element binding protein phosphorylation in dorsal root ganglion neurons

Actions of gonadal steroids have not been widely investigated in the peripheral nervous system, although many dorsal root ganglion (DRG) and autonomic pelvic ganglion (PG) neurons express estrogen receptors (ERs). We have studied the effects of 17β-estradiol exposure on cultured DRG and PG neurons from adult rats. Western blotting analysis of DRG extracts detected phosphorylation of ERK1 and ERK2 (extracellular signal-regulated kinases) that peaked 10 min after exposure to 17β-estradiol. These extracts contain both neurons and glia; therefore, to determine if this response occurred in DRG neurons, we developed an immunocytochemical method to specifically measure activation in individual neurons. These measurements showed that estradiol increased phosphorylation of CREB (cyclic AMP response-element binding protein), which was consistently blocked by the ERK pathway inhibitor PD98059 but not by the inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY294002. 17β-Estradiol activation of CREB in DRG neurons was reduced by the ER antagonist, ICI182780. In contrast, in PG neurons estradiol did not affect CREB phosphorylation, highlighting a difference in E2 responses in different populations of peripheral neurons. This study has shown that estrogens can rapidly activate signaling pathways associated with CREB-mediated transcriptional regulation in sensory neurons. As these pathways also mediate many effects of neurotrophic factors, changes in estrogen levels (e.g. during puberty, pregnancy or menopause) could have broad-ranging genomic and non-genomic actions on urogenital pain sensation and reflex pathways.

Substance P in the uterine cervix, dorsal root ganglia and spinal cord during pregnancy and the effect of estrogen on SP synthesis

Prior to parturition the non-pliable uterine cervix undergoes a ripening process (“softens” and dilates) to allow a timely passage of the fetus at term. The exact mechanism(s) triggering and involved in cervical ripening are unknown, though evidence for a role for sensory neurons and their contained neuropeptides is emerging. Moreover, an apparent increase in neuropeptide immunoreactive nerves occurs in the cervix during pregnancy, maternal serum estrogen levels rise at term and uterine cervix-related L6-S1 dorsal root ganglia (DRG) sensory neurons express estrogen receptor (ER) and neuropeptides. Thus, we sought to test the hypothesis that the neuropeptide substance P (SP) changes biosynthesis and release over pregnancy, that estrogen, acting via the ER pathway, increases synthesis of SP in DRG, and that SP is utilized in cervical ripening at late pregnancy. Using immunohistochemistry, in situ hybridization, reverse transcriptase–polymerase chain reaction (RT–PCR) and radioimmunoassay (RIA), we investigated coexpression of ER-α/β and SP; differential expression of ER-α and -β mRNA in DRG neurons; SP synthesis in DRG; and changes in SP concentration in the cervix, DRG and spinal cord over pregnancy. In addition, the effect of exogenous estrogen on SP synthesis in L6-S1 DRG of ovariectomized rats was examined. SP-immunoreactive neurons expressed ER-α and ER-β. SP synthesis (expressed as β-PPT mRNA label) was prominent in small DRG neurons. SP concentration increased in the L6-S1 DRG and spinal cord segments, with a peak at Day 20 of gestation, but decreased in the cervix during the first two trimesters, with a rise over the last trimester to Day 10 levels. SP and ER-α mRNA synthesis increased in DRG over pregnancy but ER-β mRNA levels were largely unchanged. When ovariectomized rats were treated with exogenous estrogen, SP mRNA synthesis in the DRG increased in a dose-related manner, an effect blocked by ER blocker ICI 182 780. From these results, we postulate that synthesis of SP in L6-S1 DRG and utilization in the cervix increase over pregnancy and this synthesis is under influence of the estrogen–ER system, most likely ER-α. We postulate that SP may play a role in cervical ripening and, consequently in the birth process.