Minireview: Neural Signaling of Estradiol in the Hypothalamus

Many of the actions of estradiol in the central nervous system (CNS) are mediated via intracellular estrogen receptors (ERs)/transcription factors that interact with steroid response elements on target genes. In addition, there is compelling evidence for membrane-associated ERs in hypothalamic and other CNS neuron; but it is not well understood how estradiol signals via membrane-associated receptors and how these signals impact neuronal excitability and physiological functions. It has been known for some time that estradiol can rapidly alter neuronal activity within seconds, indicating that some cellular effects can occur via membrane initiated events. Thus, estradiol can affect second messenger systems, including calcium mobilization and a plethora of kinases to alter cell excitability and even gene transcription in hypothalamic neurons. Therefore, this minireview will summarize our current knowledge of rapid membrane-initiated and intracellular signaling by estradiol in the hypothalamus, the nature of receptors involved, and how they contribute not only to control of reproduction but other vital homeostatic functions.

Estrogen Neurobiology

17β-estradiol (E2) modulates hypothalamic neuronal excitability that ultimately regulates reproduction, energy balance, temperature, circadian rhythms, and stress. In addition, E2 is involved in neuronal synaptic plasticity in the hippocampus, striatum, and cerebellum (1–3). Obviously, E2 signaling in the hypothalamus is the quintessential function that controls reproduction (4). In females, E2 signaling in the hypothalamus is the basis of positive and negative feedback within the hypothalamic-pituitary-ovarian axis. The endocrine status of gonads is communicated to the brain by circulating E2 that activates hypothalamic circuits that regulate ovulation. E2 both inhibits and stimulates the release of GnRH and LH, as well as FSH, and stimulates sexual behavior. E2 binds to and activates the classical ERα and ERβ but also G protein-coupled metabotropic receptors.

Classically, ERs were defined by their ability to bind estrogens and elicit a specific response (5). They were considered cytosolic receptors that upon E2 binding underwent a conformational change and translocation to the nucleus, where they interacted with DNA to regulate the expression of targeted genes. Subsequently, ERα (ESR1) and ERβ (ESR2) were cloned in the 1980s and 1990s, respectively (6, 7). Although they are the product of different genes, ERα and ERβ share a similar modular structure that binds E2 and have significant sequence homology, especially in their DNA and ligand binding domains. Also, ERα and ERβ interact with other transcription factors, such as Fos and Jun, which bind DNA at the activator protein-1 (AP-1) site, to regulate transcription independent of estrogen-response elements (EREs) (8).

Another parallel line of research developed in the 1970s that implicated E2 in rapid, nongenomic actions in numerous neuronal and nonneuronal cells: E2 membrane signaling rapidly increased levels of cAMP in the uterus (9), altered firing of hypothalamic neurons within seconds (10), and the release of neuropeptides (11). However, the concept of “rapid” nongenomic effects for estrogen signaling was foreign to endocrinologists. Although E2 elicited effects on hypothalamic and hippocampal neurons at subnanomolar concentrations, there did not appear to be identifiable steroid receptors associated with the plasma membrane for mediating these rapid actions (12, 13). This changed in the 1990s, when membrane localization of ERα was documented in pituitary cells and primary cultures of hippocampal CA1 neurons (14, 15). Moreover, Razandi et al (16) discovered that nuclear and membrane receptors were encoded by the same ER genes, and ERα and ERβ were shown to complex with G protein signaling cascades. In addition, several groups had identified membrane ERs (mERs) that were not derived from ERα or ERβ transcripts (17–19), including a bona fide G protein-coupled receptor (GPCR), GPR30/GPER1 (20, 21). It was evident from the investigation of “nongenomic” signaling that although some of these signaling cascades initiated at the membrane were tied to rapid membrane effects on ion channel activity, others led to the regulation of gene transcription, similar to the membrane-to-nucleus signaling described for many neurotransmitters (22). In this light, it has been more accurate to differentiate between membrane-initiated signaling and nuclear-initiated signaling when discussing hormone actions in neurons and nonneural cells (23). Therefore, this minireview will summarize the role of mERs in hypothalamic functions, keeping in mind that similar membrane-initiated actions of E2 have been documented in other brain structures, such as the hippocampus, striatum and cerebellum, CNS structures involved in cognition and motor functions, respectively (1–3).

Nuclear-Initiated Signaling of E2

Early studies used ³H-E2 to explore binding sites in the brain, and these studies revealed that estradiol-concentrating neurons were localized in hypothalamic regions, including the preoptic area (POA), periventricular nucleus (PV), and arcuate nucleus (24–28). Once ERα and ERβ were cloned, their distribution was thoroughly elucidated using in situ hybridization and/or immunocytochemistry (29–39). ERα is robustly expressed in regions such as the POA, bed nucleus stria terminalis, amygdala, PV, ventrolateral part of the ventromedial nucleus of the hypothalamus, and the arcuate nucleus. ERβ is found in many of the same regions, but is more highly expressed in the bed nucleus stria terminalis, POA, PV of the hypothalamus, and supraoptic nuclei, with some notable species differences (32, 33, 39–41). ERα and ERβ are also found in other brain regions, including the cortex, hippocampus, midbrain, and striatum (32, 42). Colocalization studies have identified ERα in hypothalamic neurons containing gamma-Aminobutyric acid (GABA), neurotensin, somatostatin, galanin, dopamine, norepinephrine, neuropeptide Y (NPY), proopiomelanocortin (POMC), and kisspeptin (Kiss1) (33, 43–50). ERβ is expressed in different populations of hypothalamic neurons: GnRH, vasopressin, oxytocin, and nociceptin/orphanin FQ, as well as in midbrain serotonin neurons (51–60). ERα and ERβ are colocalized in neurons expressing corticotropin-releasing hormone and IGF-1, as well as in subpopulations of unidentified hypothalamic neurons (37, 52, 61, 62).

The nuclear-initiated signaling of estradiol via ERα and ERβ exerts diverse effects in a number of tissues that involves gene stimulation as well as gene repression (63–68). In general, the “classical” signaling pathway of E2 involves steroid-dependent formation of nuclear ER homo- or heterodimers and the subsequent binding of this complex with a unique DNA sequence known as an ERE, in E2-responsive gene promoters and enhancers (69–71).

However, many genes in the brain that are estrogen-responsive do not appear to contain ERE sequences (71, 72). There is compelling evidence that ERα and ERβ can regulate transcription of some of these “estrogen-responsive” genes by interacting with other DNA-bound transcription factors, such as specificity protein-1 and AP-1, rather than binding directly to DNA (71, 73, 74). In contrast to ERα, the ligand-induced responses with ERβ at an AP-1 site illustrate the negative transcriptional regulation by estrogens and strong positive regulation by ER antagonists like ICI 164384 (73). In addition, Kiss1 mRNA is differentially regulated by E2 in the anteroventral periventricular preoptic area and arcuate nucleus. Although the positive E2 regulation of Kiss1 mRNA expression in the AVPV is dependent on an ERE-binding site, the down-regulation of Kiss1 mRNA in the arcuate nucleus is via an ERE-independent mechanism (75). Therefore, there are potentially multiple mechanisms for differential regulation of gene expression by E2 via nuclear-initiated signaling.

Membrane-Initiated Signaling of E2

Selective membrane binding sites for E2 were first identified on endometrial cells (76, 77), and later studies revealed relatively high affinity, specific binding of [³H]-E2 to synaptosomal membranes prepared from the adult rat brain (78). The CNS binding was later corroborated using the membrane impermeant E2–6-[¹²⁵I] conjugated to BSA (79). Furthermore, competition-binding assays of synaptosomal membranes showed that the hypothalamus exhibited a relatively high-affinity (3nM) binding site for E2 and somewhat lower affinity binding sites in the olfactory bulb and cerebellum (80, 81). The stereospecificity of the binding was demonstrated by displacement of the radiolabeled E2 with cold E2 or E2-BSA, but not by 17α-estradiol or 17α-estradiol-BSA even at micromolar concentrations (80).

In parallel electrophysiological studies, E2 was shown to have acute, rapid membrane-initiated signaling actions in many CNS structures, including the hypothalamus (10, 82–89). Three decades ago, the nature and physiological significance of these actions were a matter of debate, but it is now generally accepted that some of the actions of E2 are too fast to be attributed to the classical nuclear-initiated steroid signaling of ERα or ERβ. We now know that ERα and ERβ can associate with signaling complexes in the plasma membrane (16, 90–94). In addition, many of the rapid effects of E2 can be induced by selective ERα or ERβ ligands, antagonized by the ER antagonist ICI 182780, and abrogated in animals bearing mutations in ERα and/or ERβ genes (64, 90, 95–99). However, it is also evident that E2 can activate bona fide GPCRs, the most notable GPR30 and a putative Gαq-coupled mER (Gαq-mER) (18, 19, 100–105).

Over the years, evidence has been generated in the support of a novel Gαq-mER. Intracellular sharp electrode and whole-cell patch recording from guinea pig and mouse hypothalamic slices were used to characterize this Gαq-mER (13, 18, 19). These 2 independent electrophysiological methods established that E2 acts rapidly and stereospecifically within physiologically relevant concentrations to significantly reduce the potency of μ-opioid and GABA_B agonists (ie, heterologous desensitization) to activate G protein-coupled inwardly rectifying K⁺ (GIRK) channels (Figure 1) (13, 18). Estrogenic desensitization of μ-opioid and GABA_B receptors was mimicked by stimulation of adenylyl cyclase with forskolin or by direct protein kinase A (PKA) activation with the nonhydrolyzable cAMP analog Sp-Cyclic 3′,5′-hydrogen phosphorothioate adenosine hydrate, in a concentration-dependent manner (13, 18). Furthermore, selective PKA antagonists blocked the effects of E2. As predicted from the literature on desensitization of GPCRs (106), PKA is downstream in a signaling cascade that is initiated by a Gαq-mER that is linked to activation of phospholipase C (PLC)-protein kinase C (PKC)-PKA (18, 19). E2 does not alter the affinity of the μ-opioid and GABA_B ligands for their respective receptors (107). Furthermore, the ER antagonists ICI 164384 and ICI 182780 block the actions of E2 with subnanomolar affinity (K_i = 0.5nM), which is similar to K_i for antagonism of ERα (13, 108). These pharmacological findings clearly argue for a G protein-coupled membrane receptor with high selectivity for E2.

Figure 1.

Cellular actions of E2 in POMC neurons. Schematic overview of the E2-mediated modulation of Gαi,o-coupled (μ-opioid and GABA_B) receptors via a membrane-associated receptor (mER) in hypothalamic POMC neurons. E2 binds to a mER that is Gαq-coupled to activate phospholipase C (PLC) and catalyzes the hydrolysis of membrane-bound phosphatidylinositol 4,5-biphosphate (PIP₂) to inositol 1,4,5 triphosphate (IP₃) and diacylglycerol (DAG). Calcium is released from intracellular stores (endoplasmic reticulum) by IP₃, and DAG activates PKCδ. Through phosphorylation, adenylyl cyclase VII (AC VII) activity is up-regulated by PKC. The generation of cAMP activates protein kinase A (PKA), which can uncouple μ-opioid (μ) and GABA_B receptors from their signaling pathway through phosphorylation of a downstream effector molecule (eg, G protein-coupled, inwardly rectifying K⁺, GIRK, channels). PKA can also potentially phosphorylate other channels (eg, canonical transient receptor potential [TRPC] channels) to alter their function (data not shown). In addition, the membrane-initiated E2 signaling through PKA can phosphorylate cAMP-responsive element binding protein (pCREB), which can alter gene transcription through its interaction with the cAMP response element (CRE). Therefore, the rapid membrane-initiated signaling can alter gene expression in an ERE-independent fashion.

The results from these early physiological and pharmacological experiments led to the design of a synthetic diphenylacrylamide compound called STX (18), which is structurally similar to 4-OH tamoxifen, for selectively targeting the Gαq-mER signaling pathway (18). As predicted, STX has greater affinity (∼20-fold) for the Gαq-mER than E2 and most importantly does not bind to ERα or ERβ (19, 109). Furthermore, both STX and E2 activate the Gαq signaling pathway in POMC neurons in mice lacking both ERα and ERβ and in GPR30 knockout mice (19, 110).

Although somewhat controversial as a stand-alone ER (111), some of the effects of E2 in the CNS are thought to be mediated via GPR30 (103, 105, 112). Initial studies showed specific binding of E2 to membranes isolated from ERα−, ERβ−, and GPR30+ (SKBR3) breast cancer cells and E2-mediated stimulation of a Gαs-coupled signaling pathway (113). The signaling pathway is somewhat circuitous and involves E2 activating Gβγ-subunits that promote the release and activation of an epidermal growth factor (EGF) precursor pro-heparin binding EGF (pro-HB-EGF) (20, 114, 115). The active HB-EGF binds to the EGF receptor to facilitate receptor dimerization and downstream activation of MAPK. Although GPR30 protein and mRNA have been localized in several brain regions, including the hypothalamus by immunocytochemical staining and RT-PCR, respectively, definitive binding studies on isolated neural membranes have not been done (103, 116–119). With respect to hypothalamic function, several studies have shown that GPR30 is involved in mediating rapid actions of E2 in monkey placode GnRH neurons (103) and adult mouse GnRH neurons (120) (see below). Therefore, E2 can rapidly alter neuronal excitability through ERα, ERβ, and/or novel GPCRs.

E2 Signaling in GnRH Neurons

The relatively fast (∼15 min) inhibition of GnRH and LH secretion by systemic administration of E2 to ovariectomized females is compatible with a membrane initiated signaling mechanism. In fact, years ago, it was found that guinea pig GnRH neurons are rapidly hyperpolarized by E2 via activation of an inwardly rectifying K⁺ conductance in the presence of tetrodotoxin, which blocks fast Na⁺ channel activity and isolates GnRH neurons from action potential-driven synaptic inputs (86, 121). In mice, physiological concentrations (picomolar) of E2 rapidly augment K_ATP (also of the inwardly rectifying family) channel activity to hyperpolarize GnRH neurons and inhibit their firing (104). E2 signals via a PKC-PKA pathway, and the selective Gαq-mER ligand STX is also able to mimic the effects of E2 in GnRH neurons (13, 18, 19, 104). Both the effects of E2 and STX are abrogated by the ER antagonist ICI 182780 with a K_i of 0.5nM (104, 122), which is similar to the K_i for antagonism of ERα in peripheral tissues (108). Female mice treated with an E2 implant for 4–7 days continue to exhibit an augmented K_ATP current, which indicates that the membrane-initiated signaling cascade persist over this time period (123). This would imply that membrane-initiated signaling by E2 is involved in negative feedback regulation of GnRH neurons.

In rhesus macaque vomeronasal explants (the source of immature GnRH neurons), E2 synchronizes Ca²⁺ oscillations in GnRH neurons to a periodicity of approximately 60 minutes, similar to pulsatile GnRH release in primates (124). Knockdown of ERα and ERβ do not block the E2-induced changes in Ca²⁺ oscillations (125), and nanomolar concentrations of a membrane-impermeant E2 (E2-dendrimer conjugate), the GPR30 agonist G1 or the Gαq-mER ligand STX stimulate Ca²⁺ oscillations within 10–20 minutes (103, 105, 126). Therefore, similar to E2, G1 and STX increase the frequency and synchronization of Ca²⁺ oscillations in rhesus macaque GnRH neurons (103, 105, 125). The effects of E2 (1nM) are eliminated in GnRH neurons transfected with GPR30 small interfering RNA (siRNA), confirming that E2 can signal via GPR30 (103). However, higher concentrations of E2 (10nM) are only partially blocked by GPR30 siRNA transfection, indicating that E2 is also signaling via other receptors (105). Indeed, STX (10nM) increases the Ca²⁺ oscillations, synchronization, and GnRH release from macaque vomeronasal explants, all of which are not altered by GPR30 siRNA transfection (105). Importantly, the effects of STX are blocked by the ER antagonist ICI 182780 and by a PLC inhibitor (105). This would suggest that E2 signaling via GPR30 and a putative Gαq-mER in rhesus macaque GnRH neurons rapidly increases calcium oscillations and synchronization, and stimulates GnRH release.

In mouse GnRH neurons, E2 acts postsynaptically via ERβ or presynaptically via ERα to rapidly alter GnRH neuronal excitability. Postsynaptically, high physiological (picomolar to nanomolar) concentrations of E2 or the selective ERβ ligand diarylpropionitrile enhance action potential firing by modulating intrinsic after hyperpolarizing and after depolarizing potentials via a PKA-dependent mechanism (127). In addition, E2 rapidly potentiates high-voltage-activated Ca²⁺ currents (L- and R-type Ca²⁺ channels) via ERβ and GPR30, suggesting that Ca²⁺ signaling is also a target for membrane-initiated signaling actions in GnRH neurons (120). In contrast, low physiological (10pM) concentrations of E2 or the selective ERα ligand propylpyrazole triol reduced action potential firing in GnRH neurons by attenuating excitatory GABAergic input (127). Collectively, these findings indicate that the rapid actions of E2 on GnRH neurons involve several membrane-associated receptors (ie, ERα, ERβ, GPR30, and Gαq-mER) that play a critical role in modulating GnRH neuronal excitability.

E2 Signaling and Sexual Receptivity

Female sexual receptivity in rats is controlled by E2 through a hypothalamic circuitry that encompasses the medial preoptic nucleus (MPN), the arcuate nucleus, and the ventromedial nucleus of the hypothalamus (89, 128). Although long considered a nuclear-mediated event, recent evidence indicates that membrane-initiated signaling of E2 is important for sexual behavior in female rats (see Refs. 129–131 for review). One of the more prominent pathways involves E2 activating an ERα complex that transactivates metabotropic glutamate type 1a receptors (mGluR1as) in the arcuate nucleus. mGluR1a receptors through a Gαq-mediated signaling cascade excite NPY/agouti-related peptide (AgRP) neurons, which indirectly excite POMC neurons (89). The MPN-projecting POMC neurons release the peptide β-endorphin that activates μ-opioid receptors, which subsequently internalize in MPN neurons (132–134). This transient activation/internalization of μ-opioid receptors in the MPN appears to be necessary for full expression of sexual receptivity measured 30–48 hours after E2 treatment (135). Infusion of a membrane-impermeant E2 or a mGluR1a agonist into the arcuate nucleus stimulates μ-opioid receptor internalization (93). More recently, Christensen and Micevych (136) showed that infusions of STX into arcuate nucleus activate and internalize μ-opioid receptors in the MPN and augment sexual receptivity in female rats given a “subbehavioral” dose of E2. These actions are blocked by arcuate infusions of an mGluR1a antagonist, which suggests that Gαq-mER can collaborate with metabotropic glutamate receptors to promote sexual receptivity in female rats.

E2 Signaling in POMC and NPY/AgRP Neurons

Energy homeostasis

Hypothalamic POMC and NPY/AgRP neurons reside within the arcuate nucleus and compose a critical circuit for regulating energy homeostasis (137). Selective optogenetic stimulation of NPY/AgRP neurons evokes intense feeding (138), and ablation of these neurons in adults causes starvation (139–141). Similar genetic approaches have revealed that POMC cells have the exact opposite actions in the control of energy homeostasis (19, 138, 142–145). Moreover, these 2 populations of neurons regulate feeding through sensing circulating levels of metabolic hormones, thereby altering their firing frequency and the release of peptide and/or amino acid neurotransmitters onto target neurons in the paraventricular nucleus (PVN) and other hypothalamic nuclei (137).

POMC and NPY/AgRP neurons are also sensitive to circulating estrogens. Experiments dating back 30–40 years determined that the anorectic effects of E2 in rodents are mediated through CNS sites of action because direct injections of E2 into the arcuate/ventromedial nucleus were effective to reduce food intake, body weight and increase wheel running activity in females (146–148). Additional experiments demonstrated that E2 up-regulates the expression of the peptide β-endorphin in POMC neurons in ovariectomized female guinea pigs (149, 150). Furthermore, postmortem studies showed that there is a decrease in hypothalamic β-endorphin expression associated with weight gain in postmenopausal women who abstained from hormone replacement therapy (151). More recently it has been shown that E2 signaling via ERα is a critical component in the regulation of energy homeostasis (152). In rodents, hypoestrogenic states are clearly associated with decreased activity and an increase in body weight (19, 148, 153–159). In humans, a loss-of-function mutation in ERα, reported in a case history from 1 male individual, resulted in a clear metabolic phenotype with expression of type 2 diabetes, hyperinsulinemia, and obesity (160). However, global reinstatement of an ERα that is lacking the ERE targeting domain is sufficient for “rescuing” the metabolic deficits in mice (161). These findings suggest an important role for non-ERE-mediated E2 signaling, albeit this could be via other transcriptional activity (162) or membrane-initiated signaling of ERα. Indeed, a point mutation in ERα (Cysteine 451 to Alanine), which precludes palmitoylation and membrane trafficking of ERα globally, creates an obese phenotype with excessive visceral fat deposition in mice fed a normal chow diet (163). Moreover, brain-specific knockout of ERα causes hyperphagia and hypometabolism (142, 164), and selective knockdown of ERα in POMC neurons recapitulates the hyperphagic phenotype in female mice (142). However, one must be cautious in interpreting global and conditional ERα gene deletion experiments, because ERα is a transcription factor affecting the expression of hundreds of genes important for cell signaling, and many of these genes are essential for membrane initiated actions of E2 that contribute to POMC excitability and hence control of energy homeostasis (72). With this caveat in mind, it appears that the arcuate nucleus and specifically POMC neurons are major targets for the anorectic actions of estrogens via ERα signaling, which underscores their importance in the control of energy homeostasis. In addition, POMC neurons are also involved in the rewarding aspects of food intake (165, 166).

The ability of STX to robustly mimic the rapid effects of E2 on POMC neuronal activity led to the hypothesis that the putative Gαq-mER may also have a role in the control of energy homeostasis. Peripheral administration of STX was found to mimic the effects of E2 in controlling energy homeostasis (Figure 2) (19, 167, 168). Both E2 and STX reduce food intake, body weight gain, and abdominal fat accumulation after ovariectomy. The reduced food intake was shown to correlate primarily with reduced meal frequency in both E2- and STX-treated ovariectomized guinea pigs (168).

Figure 2.

E2 and STX attenuate the body weight gain in female guinea pigs after ovariectomy. Ovariectomized female guinea pigs were given bidaily sc injections of vehicle, E2 benzoate (EB), or STX starting on day 0 (1 week after ovariectomy). Both EB and STX significantly decreased body weight gain (two-way ANOVA overall significant effect, P < .001), and post hoc Newman-Keuls analysis revealed daily significant differences between EB and vehicle-treated, and STX and oil-treated groups (**, P < .01). (Reproduced from Roepke et al [167].)

We recently discovered a complementary Gαq-mER-mediated anorexigenic pathway in NPY/AgRP neurons. E2 and the Gαq-mER ligand STX rapidly enhance (sensitize) the GABA_B receptor agonist baclofen activation of GIRK channels, and this effect is blocked by the ER antagonist ICI 182780 (169). We also identified an ERα-mediated signaling pathway that opposes the Gαq-mER signaling cascade: activating ERα with the selective agonist propylpyrazole triol rapidly suppresses GABA_B-mediated activation of GIRK channels in NPY/AgRP neurons (169). In gonadectomized mice, the “nonselective” ligand E2 can either enhance or suppress GABA_B-mediated currents (169). However, coadministering phosphatidylinositol 3 kinase (PI3K) inhibitors, specifically a selective inhibitor of p110β, results in E2 enhancing the GABA_B receptor-mediated response. Thus, ERα via PI3K attenuates (desensitizes) the GABA_B receptor-mediated response in NPY/AgRP neurons. Physiologically, the effects of E2 could depend on the relative expression of ERα vs Gαq-mER in these orexigenic cells. NPY/AgRP neurons may serve a metabolic function when Gαq-mER expression predominates, whereas they may serve a reproductive function when more ERα is expressed (Figure 3) (see Ref. 170 for review).

Figure 3.

Effects of E2 and STX on hypothalamic POMC and NPY/AgRP neurons. Summary of how E2 excites POMC neurons and inhibits NPY/AgRP neurons via a Gαq-mER-mediated signaling pathway to regulate metabolism (A). Activation of the Gαq pathway attenuates the coupling of the GABA_B receptor to GIRK channel activation in POMC neurons, thereby increasing the excitability, but enhances the GABA_B receptor activation of GIRK channels in NPY/AgRP neurons, thereby decreasing their excitability. In NPY/AgRP neurons, ERα is also associated with the membrane, and its activation leads to attenuation of the GABA_B receptor coupling to GIRK channels (B), which is hypothesized to be involved in the control of reproduction (see text for more details).

Systemic treatment with STX, similar to E2, regulates gene transcription in the arcuate nucleus, and many of the genes are involved in the control of neuronal excitability (eg, the T-type calcium channel subunit Ca_V3.1) and intracellular signaling in arcuate neurons (167). For example, the PI3K regulatory subunits are regulated by E2 and STX: PI3K p55γ mRNA is increased by E2 treatment (171) and PI3K p85α mRNA is up-regulated by STX (167). Therefore, the putative Gαq-mER may also function in the estrogenic control of energy homeostasis through direct excitation of POMC and inhibition of NPY/AgRP neurons through a Gαq signaling cascade to alter gene transcription in these anorexigenic and orexigenic neurons, respectively. Indeed, the electrophysiological effects of STX in NPY/AgRP neurons are consistent with the finding that STX down-regulates arcuate NPY mRNA expression in ovariectomized female guinea pigs (167).

What are the source of these high picomolar (K_i of ICI 164384 = 0.5nM) (13) concentrations of E2 to cause these rapid effects on POMC and NPY/AgRP neurons? Although circulating levels of E2 are in the low to high picomolar range, there is now compelling evidence that E2 (peak levels > 1000 pg/mL) is locally synthesized and released from the mediobasal hypothalamus of rhesus macaques (172). Neuroestrogen production has also been measured in the hippocampus, cerebellum, and brainstem (see Ref. 173 for review). Therefore, not only ovarian estrogens but locally produced E2 could activate the Gαq-mER signaling cascade to provide continued excitation of POMC neurons and inhibition of NPY/AgRP neurons. However, food intake is depressed during the preovulatory phase of the menstrual cycle in humans, monkeys, and guinea pigs that correlates with peak levels of circulating E2 (see Ref. 174 for review); and ovariectomy (or menopause) often leads to increased food intake and weight gain, which argues for a substantial role of ovarian E2 feedback in the hypothalamic control of energy homeostasis.

Thermoregulation

Another critical homeostatic function controlled by the hypothalamus and modulated by circulating estrogens is the maintenance of core body temperature (Tc) (175). In fact, approximately 80% of perimenopausal/postmenopausal women experience hot flashes (176), characterized by periods of sweating and peripheral vasodilation, and are often associated with increased environmental temperature (177). A hot flash can be characterized as an exaggerated heat dissipation response initiated by the preoptic temperature-sensitive neurons. Although the mechanisms generating this response are not well understood, repeated observations have found that most hot flashes are preceded by elevation in Tc independent of peripheral vasoconstriction or elevated metabolic rate (178–180). Therefore, it has been postulated that elevated Tc may serve as one trigger of menopausal hot flashes (177, 181). There is compelling evidence for a reduction in the incidence of hot flashes in hypoestrogenic females with E2 treatment (181–183). The specific cellular mechanisms underlying actions of estrogens are unknown. However, E2 lowers Tc and in addition reduces hot flashes by raising the Tc sweating threshold (181–183). The estrogenic reduction in the naloxone-induced rise in tail skin temperature of morphine-dependent ovariectomized rats has been the “industry standard” for a hot flash model (184–186). However, the morphine-dependence, naloxone-precipitated withdrawal rat model has a number of problems related to the adverse autonomic reactions independent of an elevation in core and skin temperature. But the model does highlight a potential role of opioids (eg, from POMC neurons) directly or indirectly on preoptic temperature-sensitive neurons, given that μ-receptors are highly expressed in the POA and warm-sensitive neurons are responsive to μ-opioid agonists (187–189).

We established a guinea pig “hot flash” model based on the hypothesis that the expression of vasomotor symptoms in menopausal women is due to an elevation in Tc and a reduced thermo-neutral zone in Tc (178–180). In the ovariectomized female guinea pig, both E2 and STX significantly reduce Tc compared with animals receiving vehicle injections (Figure 4) (168). Although the cellular target for E2 modulation of Tc has not been identified, one potential mechanism is the direct action of E2 on thermosensitive (GABAergic) neurons in the medial POA of the hypothalamus (190–192). Previous studies in ovariectomized female guinea pigs have shown that medial preoptic GABAergic (putative thermosensitive) neurons respond to acute E2 treatment via a Gαq-mER signaling pathway that attenuates GABA_B inhibitory input, leading to increased neuronal excitability (193). Activation of these medial preoptic GABA neurons are responsible for evoking vasomotor responses in rodents (194) underlying heat dissipation responses (ie, vasodilatation, sweating) as seen in women experiencing hot flashes.

Figure 4.

E2 and STX reduce core body temperature (Tc) of ovariectomized female guinea pigs. Similar to E2 benzoate (EB)-treated females (8 μg/kg), STX (6 mg/kg) significantly decreased Tc compared with the propylene glycol vehicle (PPG). One week after ovariectomy, females were given daily sc injections of ligands at 10 am on M, W, and F. The data are presented as the mean ± SEM for each hour of the day averaged over 3 weeks of temperature probe recordings (ie, 3 weeks of temperature probe [SubCue DataLogger] measurements were collapsed down to a 24-h period). The data were analyzed using a two-way ANOVA (P < .05, F = 4.787, df = 2) with post hoc Newman-Keuls multiple comparison tests. All data points for both STX and EB were significantly different from vehicle (P < .01) except for the STX 2 pm to 4 pm hour time points (P < .05). The solid bar above the x-axis represents lights off or nighttime. (Reproduced from Roepke et al [168].)

Bone metabolism

Recently, STX has been found to mimic E2's effects on bone density in the ovariectomized female guinea pigs (168). Although E2 has direct effects on the osteoclast/osteoblast cells involved in bone remodeling (195, 196), E2 can reduce bone loss, a hallmark of hypoestrogenic states, in part, through the preautonomic nuclei in the hypothalamus. It is known that the preautonomic PVN neurons drive sympathetic activity and are involved in bone remodeling via a β2-adrenergic receptor-dependent mechanism (197, 198). There is an increase in bone formation and a decrease in bone reabsorption in β2-adrenergic receptor knockout mice (199). Unlike wild-type mice, gonadectomy of β2-adrenergic receptor knockout mice does not alter bone mass or bone resorption parameters, indicating that increased sympathetic activity may be responsible for the bone loss in hypoestrogenic states (199). The preautonomic PVN neurons receive a robust μ-opioid receptor inhibitory input from POMC neurons (200); therefore, E2 via Gαq-mER can affect their excitability via inhibitory POMC input. We found that STX, like E2, preserves cancellous bone density after ovariectomy, which supports a potential role of this hypothalamic signaling pathway in maintenance of bone density (168). However, STX, like E2, may also have peripheral effects (eg, directly on osteoblasts), which cannot be ruled out. Certainly, the decreased PVN output would dampen the sympathetic nervous system activity that controls bone remodeling via the β2-adrenergic receptor activity in the osteoblast cells (198).

Conclusions

Based on decades of research, it is now clear that E2 can act much like a metabotropic (GPCR) neurotransmitter to alter neuronal excitability and vital autonomic functions controlled by the hypothalamus. Signals initiated by E2 at the plasma membrane can trigger multiple intracellular signaling cascades (eg, MAPK, PI3K, and PKC) that result in the phosphorylation of hundreds of proteins that ultimately affect not only cell excitability but also gene transcription. For example, E2 turns on adenylyl cyclase activity and increase cAMP levels in POMC neurons within minutes (13). cAMP activates PKA, which in turn phosphorylates K⁺ channels and/or calcium channels to alter their activity. PKA can also phosphorylate cAMP-responsive element binding protein to elicit new gene transcription. In contrast to the anorexigenic POMC neurons, engagement of the putative Gαq-mER in orexigenic NPY/AgRP neurons elicits the opposite response, ie, an increase in K⁺ channel activation by the GABA_B receptor (Figure 3) and down-regulation of NPY mRNA expression. Thus, E2 can act on hypothalamic neurons in a cell-specific manner to generate the appropriate physiological responses by eliciting a combination of rapid changes in membrane excitability accompanied by slower alterations in gene expression. A future challenge is to identify the putative Gαq-mER, its interaction with other metabotropic receptors (eg, mGluR1a) and its physiological functions not only in the hypothalamus but throughout the CNS.