In rats, female sexual behavior is regulated by a well defined limbic–hypothalamic circuit that integrates sensory and hormonal information. Estradiol activation of this circuit results in μ-opioid receptor (MOR) internalization in the medial preoptic nucleus, an important step for full expression of sexual receptivity. Estradiol acts through both membrane and intracellular receptors to influence neuronal activity and behavior, yet the mechanism(s) and physiological significance of estradiol-mediated membrane responses in vivo have remained elusive. Recent in vitro evidence found that stimulation of membrane-associated estrogen receptor-α (ERα) led to activation of metabotropic glutamate receptor 1a (mGluR1a). Furthermore, mGluR1a signaling was responsible for the observed downstream effects of estradiol. Here we present data that show that ERα and mGluR1a directly interact to mediate a rapid estradiol-induced activation of MOR in the medial preoptic nucleus, leading to female sexual receptivity. In addition, blockade of mGluR1a in the arcuate nucleus of the hypothalamus resulted in a significant attenuation of estradiol-induced MOR internalization, leading to diminished female sexual behavior. These results link membrane-initiated estradiol actions to neural events modulating behavior, demonstrating the physiological importance of ERα-to-mGluR1a signaling.
In recent years it has become clear that estradiol can signal through membrane-associated estrogen receptors (ERs). These actions are separate from classical estradiol actions mediated by intracellular ERs, which are ligand-activated transcription factors that regulate gene expression and protein synthesis. Nuclear ER effects have a measurable time course of hours to days and play a well established role in estradiol regulation of female rat sexual receptivity. Conversely, membrane ER effects are often observed within seconds to minutes of steroid application, but their physiological significance is less well understood.
Various hypotheses suggest potential mechanisms of membrane-initiated estradiol responses (Filardo et al., 2002; Toran-Allerand et al., 2002; Qiu et al., 2003b), one of which involves estradiol binding to classical ERs at the cell surface plasma membrane (Razandi et al., 2003; Abraham et al., 2004; Chaban et al., 2004). In cultured neurons, estradiol activation of membrane ERs leads to the direct stimulation of metabotropic glutamate receptors (mGluRs), independent of glutamate activation (Boulware et al., 2005). This ER/mGluR1a interaction provides an explanation for both observed inhibitory and excitatory actions of membrane-initiated estradiol signaling. For example, the decrease of stimulated calcium flux through L-type voltage-gated calcium channels is mediated by mGluR2/3. In contrast, the activation of phospholipase C, inositol trisphosphate receptor, and mitogen-activated protein kinase (MAPK) signaling, resulting in cAMP response element-binding protein (CREB) phosphorylation, is dependent on mGluR1a. The present experiments were performed to determine whether mGluR-dependent estradiol signaling, demonstrated in vitro, has a physiological role in modulating female sexual receptivity, a well established behavior that is reliant on estradiol.
The rationale for these studies is based on recent data indicating that estradiol influences sex behavior, not just through intracellular receptors, but also by acting at the membrane surface (Kow and Pfaff, 2004). Little is known, however, about the underlying signaling events mediating these membrane-initiated responses in vivo. Sexual receptivity in female rats is regulated by estradiol acting on a limbic–hypothalamic circuit that integrates sensory, endocrine, and metabolic information (Micevych and Sinchak, 2006). We focused on a portion of this circuit that originates in the arcuate nucleus of the hypothalamus (ARH) and is regulated by estradiol rapidly acting through ERα (Micevych et al., 2003; Mills et al., 2004). Estradiol control of the lordosis reflex involves the activation and internalization of μ-opioid receptors (MORs) in the medial preoptic nucleus (Eckersell et al., 1998; Sinchak and Micevych, 2001). MOR activation/internalization is dependent on estradiol-triggered neuropeptide Y (NPY) release that stimulates NPY-Y1 receptors on β-endorphin (β-END) neurons in the ARH (Mills et al., 2004; Sinchak et al., 2006). It is these NPY-receptive ARH neurons that project to the medial preoptic nucleus, release β-END, and stimulate MOR, leading to their internalization. MOR activation in the medial preoptic nucleus is needed for the complete display of lordosis behavior (Torii et al., 1996; Sinchak et al., 2005), underlining the importance of the projection from ARH to medial preoptic nucleus.
Materials and Methods
Male and ovariectomized female (200–250 g) Long–Evans rats were purchased from Charles River (Portage, MI). Females were bilaterally ovariectomized by the supplier. After arrival, male and female rats were housed in a climate-controlled room, two per cage in a 12 h light/dark cycle room (lights on at 6:00 A.M.) and provided food and water ad libitum. All experimental procedures were approved by the Chancellor's Animal Research Committee at the University of California, Los Angeles.
Guide cannula implantation surgery.
To administer the appropriate drugs to the ARH, female rats were anesthetized with isoflurane (2–3% in equal parts oxygen and nitrous oxide) and fixed in a stereotaxic frame. Bilateral guide cannulas (24 gauge; Plastics One, Roanoke, VA) directed at the ARH (coordinates from bregma: anterior, −2.0 mm; lateral, 0.8 mm; ventral, −6.9 mm from dura; tooth bar, −3.3 mm) were implanted using standard stereotaxic procedures. The cannulas were secured to the skull with dental acrylic and stainless steel bone screws. Stylets were placed in the guide cannulas that protruded <0.5 mm beyond the opening of the guide cannulas. Animals were singly housed after surgery, received oral antibiotics (trimethoprim and sulfamethoxazole; 0.4 mg/ml; Hi-Tech Pharmacal, Amityville, NY) dissolved in the drinking water, and were allowed to recover 7 d before behavioral testing.
Steroid priming and behavioral test design.
As appropriate, 17β-estradiol benzoate (EB) was used to either elicit lordosis behavior (5 μg) or provide a sub-behavioral (2 μg) dose on which the facilitation of the behavior or MOR internalization was studied. EB dissolved in safflower oil was injected subcutaneously in a total volume of 0.1 ml. Each female received sequential injections of EB 3 h before the dark phase once per 4 d cycle. Behavioral testing began 30 h after EB injection, 3 h into the dark phase. Female rats were tested for sexual receptivity during the second steroid treatment cycle after surgery to confirm responsiveness to steroids. To study the effect of blocking or activating mGluR1a on lordosis behavior, for the third steroid treatment after cannula implantation, females were first microinjected with a selective mGluR1a antagonist, (S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385; Tocris Bioscience, Ellisville, MO), or a selective mGluR1 agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG; Tocris Bioscience), followed by 5 μg of EB 30 min later.
To measure sexual receptivity, each female was placed in a Plexiglas testing arena with a stud male, where males were acclimatized to the arenas for at least 30 min before testing. Males were allowed to vigorously mount females 10 times. The number of times that the female displayed lordosis (lifting of the head, arching of the back, movement of the tail to one side) when mounted by a male was recorded. For each female, a lordosis quotient (LQ) was calculated (number of lordosis displays/number of mounts × 100) as a measure of sexual receptivity. Data were analyzed by two-way ANOVA and Student–Newman–Keuls (SNK) post hoc analysis, in which p < 0.05 was considered significant.
mGluR1a antagonist LY367385 (50 nmol) and mGluR1a agonist DHPG (50 or 200 nmol) were dissolved in artificial CSF (aCSF) vehicle. E6-biotin (Steraloids, Newport, RI) and biotin (50 nmol; Sigma-Aldrich, St. Louis, MO) were dissolved in DMSO. Microinjections were done with an infusion pump (Harvard Apparatus, Holliston, MA) at rate of 1.0 μl/min. The microinjection needle (28 gauge) protruded 2 mm beyond the opening of the cannula and was allowed to remain in place for 1 min after injection to allow for diffusion of drug treatment or aCSF control vehicle from the injector. After microinjection, obturators were reinserted into the guide cannulas, and animals were returned to their home cage until time of behavioral testing.
Confirmation of guide cannula placement.
At the end of the behavioral series, animals were anesthetized with sodium pentobarbital (100 mg/kg) and transcardially perfused with chilled 0.9% saline, followed with 4% paraformaldehyde dissolved in 0.2 m Sorenson's phosphate buffer, pH 7.4. Brains were removed and placed into fixative overnight at 4°C. The fixative was replaced with 20% sucrose in phosphate buffer to cryoprotect the tissue. Brains were blocked, sectioned (20 μm) on a cryostat (Zeiss, Thornwood, NY), and collected into a chamber filled with PBS. Sections were mounted onto SuperFrost/Plus slides (Fisher, Pittsburgh, PA), stained with thionin, dehydrated, and coverslipped with Permount (Fisher). Injection sites were mapped and verified with bright-field illumination. In nine rats, the cannulas were not positioned in the ARH (located in, above, or lateral to the ventromedial nucleus of the hypothalamus) or the microinfusion compromised the wall of the third ventricle. None of the LY367385-infused animals were misses; all of the misplaced cannulas were in the DHPG experiments. There is no consistent pattern of MOR internalization when cannulas were outside the ARH.
Animals were perfused as described above 30 min after estradiol injection and immediately processed for immunohistochemistry. For MOR internalization, rabbit primary antibodies directed against MOR (1:24,000; Neuromics, Minneapolis, MN) were used. To look at estradiol activation of intracellular signaling pathways, a rabbit antibody directed against pCREB (1:4000; Millipore, Billerica, MA) was used. Sections that were processed for fluorescence were incubated in blocking buffer (Tyramide Signal Amplification kit; NEN Life Science Products, Boston, MA) and then in biotin-conjugated goat anti-rabbit IgG (1:200; Vector Laboratories, Burlingame, CA) for 1 h. Tissue was then washed in Tris-buffered saline and incubated in streptavidin-horseradish peroxidase (1:100; NEN Life Science Products) for 30 min, washed, and then incubated for 5 min in fluorescein-conjugated tyramide or tetramethylrhodamine (Tyramide Signal Amplification kit; 1:50; NEN Life Science Products). Sections were washed again in 0.1 m Tris buffer and mounted on SuperFrost/Plus slides. Mounted sections were air dried and coverslipped using Vectashield mounting medium (Vector Laboratories).
To determine whether ERα was expressed in cells with mGluR1a, 20 μm sections through female ARH were collected and processed as described above. ARH sections were first incubated with a rabbit mGluR1 primary antibody (1:2500; Millipore) overnight at 4°C and then processed as previously described with a Fluorescein Tyramide Signal Amplification kit. After incubation with fluorescein tyramide, sections were washed in 0.1 m Tris-buffered saline and incubated with an avidin/biotin-blocking kit (Vector Laboratories) before incubation with a rabbit ERα primary antibody (C1335; 1:10,000; Millipore) for 48 h. Tissue was then washed in 0.1 m Tris-buffered saline before incubation with rhodamine [tetramethylrhodamine isothiocyanate (TRITC)]-conjugated AffiniPure goat anti-rabbit IgG (1:200; Jackson ImmunoResearch, West Grove, PA) for 2 h. Sections were washed again in Tris buffer and mounted onto SuperFrost/Plus slides and coverslipped. For quantification of the number of immunofluorescent mGluR1a and ERα, cells were counted from six sections per animal. Cells were considered colocalization if both mGluR and ERα immunoreactivity were present.
All immunohistochemically labeled sections were examined with a Zeiss Axioskop 2 equipped with epifluorescent illumination, Axiocam CCD camera, and digital image analysis system to determine colocalization and verify internalization patterns. Fluorescein (FITC) and rhodamine (TRITC) were imaged with a 488 and 550 nm emission filter and a 515–540 nm and bandpass filter, respectively. Images were adjusted for brightness and contrast using the Zeiss LSM-PC and Adobe (San Jose, CA) Photoshop (version 6.0) programs.
To obtain an estimate of relative internalization, the area of MOR immunoreactivity in the dorsal aspect of the medial preoptic nucleus in every fourth section was estimated using the National Institutes of Health (Bethesda, MD) ImageJ software (version 1.32j). Images taken at a magnification of 360× were converted to grayscale and adjusted for brightness and contrast in Adobe Photoshop (version 6.0). ImageJ was set to the “pixel inverter” function and calibrated. In each picture, a circle of ∼60 μm diameter was placed on the medial preoptic nucleus and then on another area negative for MOR immunoreactivity. A measurement of the staining was taken in the medial preoptic nucleus and subtracted from the measurement of the background staining, yielding the intensity of MOR immunostaining alone in the medial preoptic nucleus. Studies in our laboratory indicate that internalized MOR immunostaining can be visualized as an increase in MOR immunofluorescence. This has been correlated with our previous method of examining changes in density of MOR-positive processes and has been used to determine the time course and intensity of estradiol-induced activation of MOR in the limbic–hypothalamic circuit (Sinchak and Micevych, 2003; Mills et al., 2004). Results were compared by two-way ANOVA and SNK post hoc test analysis, in which p < 0.05 is considered significant. For quantification of pCREB-immunoreactive cells, six sections were counted per animal and compared across the experimental conditions using a t test with differences at the p < 0.05 level considered significant.
Coimmunoprecipitation was performed using the Catch and Release version 2.0 Reversible Immunoprecipitation System (Millipore). Briefly, immunoprecipitation (IP) samples from lysed HEK-293 cells transfected with mGluR1a and either enhanced green fluorescent protein (EGFP)-tagged ERα or EGFP were incubated with either the polyclonal mGluR1a antibody (4 μg; BD Biosciences PharMingen, San Diego, CA) or the normal rabbit negative control (4 μg; Millipore) in spin columns containing 0.5 ml of IP capture resin for 1 h at 4°C with gentle agitation. Columns were washed three times, followed by sample elution. The eluates were then separated on an SDS-polyacrylamide gel (4–12%) and transferred to a nitrocellulose membrane. Membranes were blocked and probed with the monoclonal EGFP antibody (0.4 μg/μl; Roche Diagnostics, Indianapolis, IN). An HRP-conjugated anti-mouse secondary antibody (1:12,500; Pierce, Rockford, IL) was used for visualization. In additional control experiments, lysate from female hippocampal cultures (9 d in vitro) was used in the immunoprecipitation steps, and ERα was detected with a polyclonal antibody (1 μg/μl; C1335; Millipore).
All data are expressed as the mean ± SEM. Mean differences between groups were determined using paired t test or one-way or two-way ANOVA followed by SNK post hoc analysis when the main effect or interaction was significant at p < 0.05. Statistical analysis was conducted using SigmaStat (version 2.03) software. The number of animals used in each experiment is specified in Results for specific experiments.
MOR internalization relies on a membrane-localized estrogen receptor
To test for the involvement of membrane ERα in regulating the lordosis circuit, E6-biotin, a membrane-impermeable form of estradiol, was injected into the ARH to demonstrate that estradiol acts at the surface of the cell and does not need to penetrate the cell to induce MOR internalization. E6-biotin significantly increased the level of MOR internalization in the medial preoptic nucleus compared with biotin alone (Fig. 1) (one-way ANOVA, p = 0.02; df = 19; F = 9.276; SNK, p < 0.05; p = 3; q = 5.826; n = 6–7 animals per group). Blocking ERs with ICI182,780 (ICI) prevented estradiol-induced MOR internalization in the medial preoptic nucleus (aCSF plus EB, 96.19 ± 8.66; ICI plus EB, 53.84 ± 4.13; paired t test, p = 0.008; t = 3.405; df = 9) indicating that estradiol-induced MOR activation is a result of the rapid actions of estradiol at the cell membrane.
mGluR1a and ERα interact in neurons of the ARH
Colocalization of mGluR1a and ERα neurons of the ARH was done immunohistochemically. mGluR1a immunoreactivity was localized in 23% of ERα-immunoreactive neurons in the ARH (Fig. 2 A–D). Similarly, 22% of mGluR1a-immunoreactive neurons in the ARH were ERα positive. These results highlight a population of neurons in the ARH in which ERα may directly interact with mGluR1a.
To test whether ERα can interact with mGluR1a, coimmunoprecipitation experiments were performed. Protein was isolated from HEK-293 cells transfected with mGluR1a and either EGFP-ERα or EGFP. EGFP-tagged ERα was used to take advantage of the specificity of EGFP antibodies. As shown in Figure 2E, mGluR1a coimmunoprecipitated EGFP-ERα. Furthermore, mGluR1a failed to coimmunoprecipitate with EGFP (Fig. 2F), signifying that coimmunoprecipitation of EGFP-ERα with mGluR1a was attributable to mGluR1a interacting with ERα and not EGFP. To verify that ERα could interact with mGluR1a in neurons, we performed additional coimmunoprecipitation experiments using cultured female hippocampal neurons, the preparation in which ERα-to-mGluR signaling was initially described. As with the HEK-293 cells, ERα coimmunoprecipitated with mGluR1a (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Together, these findings strongly suggest a potential physical linkage between ERα and mGluR1a that underlies the ability of membrane-localized ERα to stimulate mGluR1a signaling.
Activation of MOR in the medial preoptic area
To further understand whether estradiol-induced MOR internalization in the medial preoptic nucleus is dependent on ERα/mGluR1a signaling in the ARH, the selective mGluR1a antagonist LY367385 was used to pharmacologically block the actions of mGluR1a. Infusion of LY367385 directly into the ARH 30 min before treating with EB (5 μg/0.1 ml) or vehicle (0.1 ml of safflower oil, s.c.) significantly attenuated EB-induced MOR internalization in the medial preoptic nucleus (Fig. 3 A–E) (two-way ANOVA, drug, p = 0.001; df = 1,17; F = 14.77; steroid, p = 0.128; df = 1,17; F = 2.56; drug × steroid interaction, p = 0.039; df = 1,20; F = 5.01; SNK, p < 0.001; p = 2; q = 5.72; n = 5–8 animals per group).
To determine whether stimulation of the mGluR1a mimicked the estradiol activation of the lordosis-regulating circuit, the selective mGluR1a agonist DHPG (200 nmol) was infused into the ARH 30 min before injections of EB (2 μg/0.1 ml) or vehicle (0.1 ml of safflower oil, s.c.). Activating mGluR1a with DHPG in the absence of EB induced MOR internalization in the medial preoptic nucleus (Fig. 3F) (two-way ANOVA, drug × steroid interaction, p = 0.033; df = 1,16; F = 5.69; SNK, p = 0.006; p = 2; q = 4.6; n = 5–6 animals per group). This level of internalization was similar to that obtained with estradiol alone or estradiol and DHPG, suggesting that both estradiol and DHPG were acting through the same signaling pathway. To determine whether ICI182,780 acted on mGluR1a, ovariectomized rats were microinjected with ICI182,780 into the ARH 30 min before receiving DHPG. ICI182,780 did not block the DHPG-induced MOR internalization (ICI plus oil, 56.92 ± 6.6; ICI plus DHPG, 69.66 ± 0.86; one-way ANOVA on ranks, H = 7.269; df = 2; p = 0.016; SNK, diff of ranks = 18; q = 3.674; p < 0.05; n = 4 animals per group). These data also suggest that activation of ER precedes that of the mGluR1a. Together, these data indicate that mGluR1a is required for estradiol activation of intracellular signaling that mediates MOR internalization/activation in the medial preoptic nucleus.
It was previously shown that estradiol acting through mGluR1a triggers mitogen-activated protein kinase-dependent CREB phosphorylation in hippocampal cultures (Boulware et al., 2005). This is a pathway through which membrane actions of estradiol may regulate gene transcription. To determine whether this pathway was being activated, we next evaluated in vivo whether estradiol would increase the numbers of phosphorylated CREB-immunoreactive cells in the ARH. This is a further demonstration of membrane estradiol signaling in the ARH and the importance of ER/mGluR1a signaling. Consistent with the hypothesis, after estradiol treatment, the number of phosphoCREB-immunoreactive neurons in the ARH was increased compared with the oil-treated group (t test, p = 0.02; t = 3.16; df = 6; n = 4). Additionally, the ability of E6-biotin to increase the numbers of phosphoCREB cells was comparable with that of EB (data not shown).
ERα/mGluR1a interactions modulate sexual receptivity
The rapid activation of the ARH-medial preoptic circuit by estradiol is essential for the full display of sexual receptivity (Torii et al., 1996; Sinchak and Micevych, 2001; Sinchak et al., 2005). To determine whether ERα-to-mGluR1a signaling was necessary for the lordosis reflex, we antagonized mGluR1a in the ARH. LY367385 was microinjected into the ARH 30 min before EB injection, and the animals were tested 30 h later to determine their LQ, a quantifiable, estradiol-dependent measure of sexual receptivity. The mGluR1a antagonist LY367385 significantly attenuated the LQ in animals treated with a dose of EB that induces lordosis (Fig. 4A) (two-way ANOVA, drug, p < 0.001; df = 1,41; F = 75.42; steroid, p < 0.001; df = 1,41; F = 19.00; drug × steroid interaction, p < 0.001; df = 1,47; F = 21.44; SNK, p < 0.001; p = 2; q = 9.82; n = 5–8). Furthermore, when the selective mGluR1a agonist DHPG was infused into the ARH, lordosis was facilitated (Fig. 4B) (two-way ANOVA, drug, p = 0.02; df = 1,44; F = 5.84; steroid, p < 0.001; df = 1,44; F = 23.48; drug × steroid interaction, p = 0.19; df = 1,44; F = 1.76; SNK, p = 0.005; p = 2; q = 4.17; n = 6–12 per group), as predicted if activation of mGluR1a is important for the display of lordosis. As a separate control, when LY367385 was microinjected into the ARH 48 h after EB in EB- or EB plus progesterone-treated animals, sexual receptivity (measured 30 min later) was neither facilitated nor inhibited, respectively (2 μg of EB, LQ = 25.0 ± 11.9; EB plus P, LQ = 94.3 ± 4.0; n = 5–6; data not shown). When cannulas were outside the ARH, DHPG did not facilitate lordosis (n = 5). These results demonstrate that activation of mGluR1a in the ARH is critical during the initial phase of estradiol signaling (Mills et al., 2004). Furthermore, these results also indicate that mGluR1a is necessary for estradiol-mediated induction of sexual receptivity but not sexual receptivity itself.
It is well documented that membrane-initiated effects of estradiol exist, but from a mechanistic standpoint, the signaling pathways have remained elusive. The present study demonstrates that specific ER/mGluR1a interactions are of fundamental importance to understanding estradiol signaling in the brain, which in turn mediates behavior. Over the past 20 years, a steady accumulation of data has demonstrated that estrogens can rapidly influence a multitude of intracellular signaling pathways. Examples include estradiol regulation of PKC (Coleman and Smith, 2001), MAPK (Watters et al., 1997; Singh et al., 1999), phospholipase C (Qiu et al., 2003a), phosphatidylinositol 3 kinase (Lee et al., 2005), intracellular calcium (Mermelstein et al., 1996; Chaban et al., 2003), and cAMP/PKA (Aronica et al., 1994; Zhou et al., 1996), all of which have been assumed to be the result of distinct intracellular signaling events. The present study in vivo and previous work in vitro (Boulware et al., 2005) indicate that ERα can activate the G-protein-coupled mGluRs to initiate intracellular signaling. The differential effects observed by estradiol across cell types may simply reflect the substitution of different classes of mGluRs that interact with membrane ERs.
Membrane-initiated estradiol effects are rapidly initiated but not necessarily short in duration. In fact, once triggered, membrane estradiol effects can be on the order of hours/days, as evidenced by their ability to regulate gene expression and protein synthesis. A clear rapid action of estradiol that appears to be important in such nongenomic to genomic signaling is the phosphorylation of CREB (Wade and Dorsa, 2003; Lee et al., 2004; Boulware et al., 2005). Estradiol has been shown to rapidly increase phosphoCREB in regions associated with the control of reproduction: the medial preoptic area, ventromedial nucleus of the hypothalamus, bed nucleus of the stria terminalis (Gu and Moss, 1996; Zhou et al., 1996; Abraham et al., 2004), and the ARH, as determined in the present study. These data provide additional support for ER/mGluR1a interactions mediating intracellular signaling in the ARH, because they recapitulate previous in vitro results (Boulware et al., 2005). Moreover, the increase in pCREB indicates that rapidly initiated membrane actions of estradiol may have long-lasting actions through linkage to regulation of gene expression and protein synthesis. Indeed, rapid membrane-initiated estradiol signaling has been shown to augment estradiol transcriptional regulation (Vasudevan et al., 2005).
It is well known that estradiol regulation of reproduction is dependent on transcriptional regulation, but Kow and Pfaff (2004) have demonstrated that estradiol action at the membrane enhances the effects of intracellular ERs and thus facilitates behavior. It is these important results, demonstrating that both membrane and intracellular ERs are important for estradiol regulation of lordosis behavior, that served as a foundation for the present study. The present results are consistent with the idea that membrane ERs participate in estradiol signaling that induces lordosis. Our studies also extend the previous studies by demonstrating that such rapid actions involve ER signaling through activation of mGluR1a receptors.
At the present time, it appears that only 23% of ER-immunoreactive neurons in the ARH also express mGluR1a. Although this is probably an underestimation because of technical issues of double immunohistochemistry, this is a substantial population. There are, however, many ER-expressing neurons that do not have mGluR1a. The reason for this has not been elucidated, but a likely possibility is that some ER-expressing neurons do not use membrane-initiated estradiol signaling. In these neurons, estradiol may act through classical intracellular ERs that are ligand-gated transcription factors. Another possibility is that estradiol may be acting through another of the estradiol-binding proteins: G-protein-coupled receptor 30 (GPR30) (Revankar et al., 2005) or ER-X (Toran-Allerand et al., 2002). The present study was based on our previous study that MOR internalization required ERα, because internalization and reproduction was disrupted in ERα knock-out but not in ERβ knock-out animals (Micevych et al., 2003; Abraham et al., 2004; Chaban and Micevych, 2005; Watermann et al., 2006). Moreover, the GPR30 agonist G1 microinjected into the ARH does not internalize MOR in the medial preoptic nucleus (our unpublished results). Together, the results indicate that these particular estradiol actions are mediated through ERα.
The importance of ERα/mGluR signaling in reproductive behavior can also account for the ability of estradiol to impact diverse intracellular pathways involved in rapid nongenomic signaling that may influence a variety of neural functions. For example, hippocampal neurons in vitro exhibited the full range of ERα/mGluR signaling (Boulware and Mermelstein, 2005), and it is in the hippocampus that estradiol influences increases in dendritic length and synaptic inputs with consequences for memory and learning (Woolley, 1998; Foy, 2001; McEwen, 2002). These and the current results support the idea that ER/mGluR signaling is used widely in the brain. Thus, an understanding of ER/mGluR signaling may be relevant not only for estradiol regulation of behavior, but for estradiol-centered therapies for neurological disorders, including neurodegenerative diseases, cognitive dysfunction, pain, epilepsy, and drug abuse (Maiese et al., 2005).
One of the signature actions of estradiol is the regulation of sexual receptivity. The estradiol regulation of this behavior was originally thought to be via intracellular receptors, and certainly this is a large component of estradiol modulation of sexual receptivity. However, the results presented here demonstrate that rapid, membrane ERα/mGluR signaling also participates in the regulation of sexual receptivity. We propose that under low systemic estradiol conditions, membrane ERs in the ARH are not activated and result in no MOR internalization/activation in the medial preoptic area. This condition produces a sexually unreceptive female (Fig. 5). However, when systemic estradiol levels reach critical levels, membrane ERs in the ARH are activated, leading to MOR internalization and subsequent full lordosis behavior (Fig. 5). According to our studies, when mGluR1a is stimulated with an agonist under low or no estradiol conditions, membrane ERs are bypassed and mGluR1a is directly activated, resulting in MOR internalization and sexual receptivity. Conversely, under high estradiol conditions, antagonizing mGluR1a blocks estradiol-induced MOR internalization and attenuates sexual behavior. By demonstrating that this classical assay of estradiol action has a rapid nongenomic component underscores the importance of ER/mGluR interactions in the brain. These data are consistent with the in vitro demonstration of ERα/mGluR1a signaling in hippocampal neurons and provide the first evidence in vivo that estradiol can signal through activation of mGluR1a. Moreover, these results indicate the necessity for defining genomic and nongenomic components of estradiol regulation of physiology and behavior. Such an analysis will be critical for identifying the exact molecular nature of estradiol signaling underlying the widespread actions of estradiol in the CNS.
This work was supported by National Institutes of Health Grants DA013185 and NS41302.
- Correspondence should be addressed to Dr. Paul Micevych, 73-078 CHS, Department of Neurobiology, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, CA 90095-1763.