Membrane-Initiated Effects of Estrogen in the Central Nervous System

32 Membrane-Initiated Effects of Estrogen in the Central Nervous System O K Rønnekleiv and M J Kelly, Oregon Health & Science University, Portland, OR, USA ß 2009 Elsevier Inc. All rights reserved.

Chapter Outline 32.1 32.2 32.2.1 32.3 32.3.1 32.3.2 32.3.3 32.4 32.4.1 32.4.2 32.4.3 32.4.4 32.4.5 32.5

Historical Perspective Estrogen Receptors and Estrogen-Binding Sites ERa and ERb Estrogen Signaling Nuclear-Initiated Signaling of Estrogen Novel ERs Membrane-Initiated Signaling of Estrogen Functional Consequences of Membrane-Initiated E2 Signaling 17b-Estradiol and Neuroprotection 17b-Estradiol and Nigrostriatal Motor Pathways 17b-Estradiol, Growth Factors, and Reproduction 17b-Estradiol and GnRH Neurosecretion Effects of 17b-Estradiol on VMH/Arcuate Neurons: Role in Regulation of Feeding Conclusions: Crosstalk between Membrane Actions and Genome Activation

References

Glossary ERa and ERb Estrogen receptors a and b are the classical estrogen receptors/transcription factors. GPCRs G-protein-coupled receptors are metabotropic receptors that have traditionally been associated with neurotransmitters. However, GPR30 is an orphan GPCR that binds 17b-estradiol. There are other membrane estrogen receptors that appear to be G-protein coupled. membrane-initiated steroid signaling Terminology adopted (vs. nongenomic) by the FASEB steroid signaling workshop to designate the activation of signaling cascades originating at the membrane. nuclear-initiated steroid signaling Terminology adopted by the FASEB steroid signaling workshop to designate the direct activation of genes by steroid receptors through nuclear events.

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32.1 Historical Perspective Evidence for rapid actions of 17b-estradiol (E2) on neurons was put forth as early as the 1960s and 1970s when a number of studies suggested that gonadal steroids modulate the electrical activity of hypothalamic neurons (Barraclough and Cross, 1963; Lincoln, 1967; Lincoln and Cross, 1967; Moss and Law, 1971; Cross and Dyer, 1970; Dyer et al., 1972; Yagi and Sawaki, 1971; Bueno and Pfaff, 1976; Yagi, 1970, 1973; Kubo et al., 1975; Whitehead and Ruf 1974; Dufy et al., 1976). Most of these studies correlated changes in the firing activity of preoptic– hypothalamic neurons with the estrous cycle of the rat, or measured firing activity following systemic injection of estrogen (estradiol benzoate (EB)) in ovariectomized rats. Although the studies were correlative in nature, they did give some indication that steroids could alter neuronal activity within a very short time frame. For example, it was noted that it took about 16min for significant changes in the firing rate of preoptic–hypothalamic neurons following a systemic (IV) bolus injection of estrogen (Yagi,

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Membrane-Initiated Effects of Estrogen in the Central Nervous System

1973). Subsequently, it was shown that microelectrophoresis of 17b-estradiol could rapidly alter neuronal firing activity in medial preoptic neurons within seconds in female rats (Kelly et al., 1976, 1977a,b, 1978a,b). The E2-induced inhibition of spontaneous activity was stereospecific (17a-estradiol had no effect) and occurred at low nanomolar concentrations. Although extracellular recording of action potentials with application of steroids via microelectrophoresis could not pinpoint the specific target cell of steroid actions to alter electrical activity, it clearly established that steroids could have rapid actions (within seconds) to alter electrophysiological activity similar to classical neurotransmitters (Kelly et al., 1977a). Using intracellular recording techniques, it was found that fast perfusion of estrogen (100pM) hyperpolarized arcuate neurons within seconds in a hypothalamic slice preparation and that this effect was reversed within minutes of washing out estrogen (Kelly et al., 1980). Similar actions of E2 were also demonstrated in ventromedial nucleus of hypothalamus (VMH) and amygdala neurons, even in the presence of protein synthesis inhibitors (Nabekura et al., 1986; Minami et al., 1990). Later, it was found that gonadotropin-releasing hormone (GnRH) neurons, identified post hoc using a combination of intracellular dye labeling and immunocytochemical staining, were a target of estrogen actions (Kelly et al., 1984). In these hypothalamic (GnRH) neurons, estrogen rapidly induced hyperpolarization via the opening of Kþ channels (Kelly et al., 1984). Selective membrane binding sites for E2 were first identified on endometrial cells (Pietras and Szego, 1977, 1979). Subsequent studies revealed relatively high affinity, specific binding of [3H]-17b-estradiol to synaptosomal membranes prepared from the adult rat brain (Towle and Sze 1983). These findings were later corroborated using the membrane impermeable 17b-estradiol-6-[125I]-BSA (Zheng and Ramirez, 1997). 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 (Ramirez et al., 1996; Ramirez and Zheng, 1996). The stereospecificity of the binding was demonstrated by displacement of the radiolabeled E2 with cold E2 or E2-BSA, but not by 17a-estradiol or 17a-estradiol-BSA even at micromolar concentrations (Ramirez et al., 1996). These biochemical data complemented the electrophysiological findings that gonadal steroid signaling could be initiated at the membrane.

32.2 Estrogen Receptors and Estrogen-Binding Sites 32.2.1

ERa and ERb

Studies utilizing 3H-estradiol to explore binding sites in the brain revealed that estradiol-concentrating neurons are localized in hypothalamic regions, including the preoptic, periventricular (PV), and arcuate nuclei (Pfaff and Keiner, 1973; Sar and Stumpf, 1975; Warembourg, 1977; Sar, 1984; Tardy and Pasqualini, 1983). Since these initial autoradiography studies, two different estrogen receptors (ERs), ERa and ERb, have been cloned and their distribution thoroughly elucidated using in situ hybridization and/or immunocytochemistry (Sar and Parikh, 1986; DonCarlos et al., 1991; Simerly et al., 1990; Shughrue et al., 1997; Laflamme et al., 1998; Osterlund et al., 2000; Gundlah et al., 2000, Shughrue and Merchenthaler, 2001; Gre´co et al., 2001; Kruijver et al., 2002, 2003). ERa is robustly expressed in regions such as the preoptic area (POA), bed nucleus stria terminalis (BNST), amygdala, PV nucleus, ventrolateral part of the VMH, and the arcuate nucleus. ERb is found in many of the same regions, but is more highly expressed in the BNST, POA, paraventricular nucleus of the hypothalamus (PVH), and supraoptic nuclei (SON), with some variation across species (Shughrue et al., 1997; Laflamme et al., 1998; Mitra et al., 2003; Kruijver et al., 2003; Warembourg and Leroy, 2004). ERa and ERb are also found in other brain regions, including the hippocampus, midbrain, cortex, diagonal band of Broca, and basal nucleus of Meynert (Shughrue et al., 1997; Merchenthaler et al., 2004). Co-localization studies have identified ERa in neurons containing g-aminobutyric acid (GABA), neurotensin, somatostatin, galanin, dopamine, neuropeptide Y (NPY), proopiomelanocortin (POMC), and metabotropic glutamate receptor 1a (Flu¨gge et al., 1986; Herbison and Theodosis, 1992; Lehman and Karsch, 1993; Herbison, 1994; Horvath et al., 1995; Skinner and Herbison, 1997; Roepke et al., 2007; Dewing et al., 2007). For the most part, ERb is expressed in different populations of neurons such as those containing GnRH, vasopressin (VP), oxytocin (OT), and nociceptin/orphanin FQ , as well as in midbrain serotonin neurons (Hrabovszky et al., 1998, 2000, 2001, 2004; Cardona-Gomez et al., 2000; Kallo et al., 2001; Skynner et al., 1999; Herbison et al., 2001; Gundlah et al., 2001; Isgor et al., 2003). In addition, ERa and ERb are both localized in neurons expressing corticotropin-releasing hormone (CRH) and

Membrane-Initiated Effects of Estrogen in the Central Nervous System

insulin-like growth factor I (IGF-I), as well as in subpopulations of unidentified hypothalamic neurons (Shughrue et al., 1998; Gre´co et al., 2001; Bao et al., 2005; Cardona-Gomez et al., 2000).

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ligand-induced responses with ERb, in contrast to ERa, at an AP-1 site illustrate the negative transcriptional regulation by estrogen and strong positive regulation by antiestrogens such as ICI 164,384 (Paech et al., 1997). This provides a mechanism for differential regulation of gene expression by estrogen.

32.3 Estrogen Signaling 32.3.1 Nuclear-Initiated Signaling of Estrogen ERs regulate cellular function through at least two signaling pathways previously broadly classified as genomic versus nongenomic (McEwen and Alves, 1999; Bjo¨rnstro¨m and Sjo¨berg, 2005). However, recently the Federation of American Societies for Experimental Biology (FASEB) steroid signaling work group suggested that membrane-initiated steroid signaling and nuclear-initiated steroid signaling are more appropriate terminologies (see Hammes and Levin (2007)). The nuclear-initiated signaling of estrogen via ERa and ERb exert diverse effects on a variety of tissues that involves gene stimulation as well as gene repression (Herbison, 1998; Couse and Korach, 1999; Nilsson et al., 2001; Etgen et al., 2001; Stossi et al., 2006; Kininis et al., 2007). In general, this classical signaling pathway of estrogen involves the steroid-dependent formation of nuclear ER homo- or heterodimers and the subsequent binding of this complex with a unique DNA sequence known as an estrogen response element (ERE), in E2-responsive gene promoters (O’Malley and Tsai, 1992; Muramatsu and Inoue, 2000; Gruber et al., 2004). The inactive ER exists in a complex of several proteins that disassociate upon ligand binding, which transforms the receptor to an active state (Couse and Korach, 1999; Gruber et al., 2004). More specifically, recruitment of other nuclear co-activator and coregulatory proteins and interactions with the transcription machinery results in transactivation of genes that contain EREs (Muramatsu and Inoue, 2000; Gruber et al., 2004). Several genes in the brain that are clearly estrogen responsive do not appear to contain ERE sequences (Malyala et al., 2004; Gruber et al., 2004). There is compelling evidence that ERa and ERb can regulate transcription of some of these estrogen-responsive genes by interacting with other DNA-bound transcription factors, such as specificity protein-1 (SP-1) and activator protein-1 (AP-1), rather than binding directly to DNA (Paech et al., 1997; Jacobson et al., 2003; Gruber et al., 2004). For example, the

32.3.2

Novel ERs

It has been known for a number of years that estrogen has acute, membrane-initiated signaling actions in the brain (for review, see Kelly and Rønnekleiv (2002), Rønnekleiv and Kelly (2005), and Bryant et al. (2006)). The nature and significance of these actions have been a matter of dispute. It is, however, now widely accepted that some of the actions of estrogen are quite rapid and cannot be attributed to the classical nuclear-initiated steroid signaling of ERa or ERb. One popular view is that both nuclear and plasma-membrane-associated ERs might be products of the same genes (Figure 1(a)) (Razandi et al., 1999; Boulware et al., 2005; Pedram et al., 2006; Szego˜ et al., 2006; Dewing et al., 2007). This belief stems primarily from the fact that many of the rapid effects of E2 can be induced by selective ERa or ERb ligands, antagonized by the ER antagonist, ICI 182,780, or are lost in animals bearing mutations in ERa and/or ERb genes (Couse and Korach, 1999; Singer et al., 1999; Dubal et al., 2001; Wade et al., 2001; Abraham et al., 2003; Boulware et al., 2005, 2007). Another view is that estrogen activates a unique membrane ER (mER) (Gu et al., 1999; Toran-Allerand, 2004, 2005; Qiu et al., 2003, 2006b). For example, in hippocampal slices E2 enhances N-methyl-D-aspartate (NMDA)-mediated excitatory postsynaptic potentials (EPSPs) and long-term potentiation (LTP) following Schaffer (collateral) fiber stimulation (Foy et al., 1999). This finding was a significant breakthrough in terms of understanding the rapid effects of estrogen in enhancing LTP in the hippocampus. Also, E2 potentiated non-NMDA (kainate)-mediated excitation of hippocampal CA1 pyramidal neurons via activation of a cAMP/protein kinase A (PKA) pathway (Gu and Moss, 1996, 1998). Interestingly, when the steroid was conjugated to bovine serum albumin (E2-BSA), it had to be applied both to the outside membrane and to the cytosolic side of the cell membrane in order to have an effect on kainate-induced currents. Based on the findings from the Moss lab, it was hypothesized that E2 activates a Gs-coupled receptor on the extracellular

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Figure 1 Cellular models of membrane-initiated signaling pathways of 17 b-estradiol (E2). (a) E2 interacts with ERa/b localized at the neuronal (plasma) membrane and activates group I and group II metabotropic glutamate receptor (mGluR1a, mGluR2/3) signaling. Activation of mGluR1a causes Gaq stimulation of phospholipase C (PLC) which leads to MAPK-induced cAMP-responsive element binding protein (CREB) phosphorylation. Activation of mGluR2/3 causes Gi/o inhibition of adenylyl cyclase (AC) and PKA which reduces the activity of L-type calcium channels and leads to attenuated CREB phosphorylation. (b) E2 activates a membrane-associated ER (mER) that is Gaq-coupled to activation of PLC that 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 PKCd. Through phosphorylation, AC activity is upregulated by PKCd. The generation of cAMP activates PKA, which can rapidly uncouple GABAB and m-opioid (m) receptors from their effector system through phosphorylation of a downstream effector molecule (e.g., G-protein-coupled inwardly rectifying Kþ [GIRK] channel). mER-mediated modulation of kinase pathways reduces the capacity of neuromodulators such as GABA and b-endorphin (b-End) to inhibit POMC neuronal excitability. ER-mediated activation of PKA can lead to phosphorylation of CREB (pCREB), which can then alter gene transcription through its interaction with the CRE. (c) E2 acts via GPR30 to promote EGF-R transactivation. This happens through a Gbg-subunit pathway that promotes intracellular tyrosine kinase-(Src-) mediated metalloproteinase (MMP)-dependent cleavage of proheparin-binding EGF (proHBEGF) and release of HB-EGF from the cell surface. EGF-R transactivation leads to Ras-dependent ERK1/2 activation and presumably CREB phosphorylation. E2 via GPR30 can also stimulate AC activity, which leads to PKA-mediated suppression of EGF-induced ERK1/2 activity. MAPK, mitogen-activated protein kinase; MEK, MAP kinase kinase; CaM, calmodulin; RSK, p90 ribosomal protein S6 kinase; CRE, cyclic AMP response element.

Membrane-Initiated Effects of Estrogen in the Central Nervous System

surface of hippocampal neurons, which operates in concert with an internal action of E2 on cyclic AMP (cAMP)-dependent phosphorylation (Gu and Moss, 1998). Importantly, these rapid actions of E2 on kainate-induced currents in hippocampal neurons were still present in animals deficient in ERa (Korach mice), suggesting a novel mechanism (receptor) for the rapid actions of E2 in the hippocampus (Gu et al., 1999). In addition, E2 and E2-BSA, when applied acutely to the hippocampus in ovariectomized animals, produced a sustained reduction of the slow IAHP in CA1 pyramidal neurons (Carrer et al., 2003). The slow IAHP is mediated by a Ca2þ-activated Kþ conductance. This provided further evidence for the involvement of an mER, although the mechanism by which E2 regulates Ca2þ influx into CA1 neurons is currently unknown. More recent studies in hippocampal CA3–CA1 neuronal cultures have revealed that E2 rapidly stimulates mitogen-activated protein kinase (MAPK)-dependent cAMP-responsive element binding protein (CREB) phosphorylation (see Figure 1(a)). In addition, E2 also decreases L-type calcium channel-mediated CREB phosphorylation, and, therefore, has both positive and negative influences on CREB activity (Boulware et al., 2005). Both effects can be induced by the membraneimpermeable E2-BSA and are inhibited by the antiestrogen ICI 182,780, which collectively suggest that an mER is involved with some of the characteristics described by Qiu et al. (2003). The positive modulation of CREB activity occurs via ER interactions with metabotropic glutamate receptor 1 (mGluR1) and the negative modulation via mGluR2/3. The type of interaction, however, is currently unknown, but potentially could be similar to that described for mER and 5HT2C receptors which have diverse but convergent signaling pathways in arcuate neurons (Qiu et al., 2007). Based on responses to the ER agonists, propylpyrazole-triol (PPT) and diarylpropionitrile (DPN), which are selective for ERa and ERb, respectively, and transfection studies with mutant ERa (Harrington et al., 2003; Lund et al., 2006; Boulware et al., 2007), the membrane-localized receptors in the hippocampus are hypothesized to be ERa and ERb (Boulware et al., 2005, 2007). However further studies, such as in ERa and/or ERb knockout (KO) mice, are needed to more clearly define these hippocampal ERs (mERs), and whether they are indeed ERa and/or ERb associated with the plasma membrane. Potential candidates for novel mERs include ER-X and the G-protein-coupled receptors (GPCRs), G-protein-coupled mER (Gq-mER) and GPR30

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(Toran-Allerand, 2005; Qiu et al., 2003, 2006b; Filardo and Thomas, 2005; Funakoshi et al., 2006). ER-X is a plasma-membrane-associated, putative ER that is enriched in caveolar-like microdomains of postnatal, but not adult, cortical membranes (Toran-Allerand et al., 1999, 2002). In organotypic explants of the developing cerebral cortex, E2 induces tyrosine phosphorylation of both extracellular signal-regulated protein kinase 1 (ERK1) and ERK2, an action very similar to a number of growth factors, including nerve growth factor (NGF) (Toran-Allerand, 2005). Interestingly, this E2-induced activation is not antagonized by the antiestrogen, ICI 182,780, and occurs in cortical explants from mice lacking ERa. In addition, this putative mER is particularly responsive to 17a-estradiol as compared to 17bestradiol (Singh et al., 1999, 2000; Toran-Allerand et al., 2002). These and other findings have led to the hypothesis that ER-X is a novel ER that is ICI insensitive (for review, see Toran-Allerand (2004, 2005)). Substantial evidence has been generated in the support of a Gq-mER. Intracellular and whole-cell recording from guinea pig and mouse hypothalamic slices have been used to characterize the mER (Lagrange et al., 1997; Qiu et al., 2003, 2006b). These studies show that E2 acts stereospecifically with physiologically relevant concentration dependence (EC50 ¼ 8 nM) to cause a significant reduction in the potency of m-opioid and GABAB agonists to activate an inwardly rectifying Kþ conductance (Lagrange et al., 1997; Qiu et al., 2003). Estrogenic modulation of m-opioid and GABAB agonists potency is mimicked either by stimulation of adenylyl cyclase with forskolin or by direct PKA activation with Sp-cAMP, in a concentrationdependent manner (Lagrange et al., 1997; Qiu et al., 2003). Furthermore, the selective mPKA antagonists KT5720 and Rp-cAMP block the effects of E2. More recent data indicate that PKA is downstream in a signaling cascade that is initiated by a Gaq-coupled mER that is linked to activation of phospholipase C (PLC)PKC-PKA (Qiu et al., 2003, 2006b). E2, however, does not compete for the m-opioid (or GABAB) receptor or alter the affinity for the receptor. Importantly, the antiestrogens ICI 164,384 or ICI 182,780 block the actions of E2 with subnanomolar affinity that is similar to ICI’s affinity (Ki) for ERa (Weatherill et al., 1988; Lagrange et al., 1997; Qiu et al., 2003). These pharmacological findings clearly define a G-protein-coupled membrane receptor for estrogen. The mER is not ERa or ERb, since it can be activated with a diphenylacrylamide compound,

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Membrane-Initiated Effects of Estrogen in the Central Nervous System

STX, that does not bind ERa or ERb (Qiu et al., 2003, 2006b). STX (and E2) selectively target a Gaq-coupled PLC-(PKC)-PKA pathway in mediating m-opioid and GABAB desensitization of hypothalamic neurons in normal animals (Figure 1(b)). Importantly, STX (and E2) can also activate the Gq signaling pathway in mice lacking both ERa and ERb (Qiu et al., 2006b). As with ER-X, definitive characterization of this Gq-mER awaits cloning of the gene. An orphan GPCR, GPR30, has also received substantial attention because it binds estrogen, albeit in cancer cells, and exhibits binding and signaling characteristics of an mER (Thomas et al., 2005). In breast cancer cells that are transfected with GPR30, estrogen activates the MAPKs, ERK1 and ERK2, and these actions are independent of ERa or ERb (Filardo, 2002; Filardo and Thomas, 2005). According to this model, E2 activates Gbg-subunits that promote the release and activation of an epidermal growth factor precursor (proheparin-binding (proHB)-EGF; Figure 1(c)). The active HB-EGF binds to the EGF receptor (ErbB) to facilitate receptor dimerization and downstream activation of ERK (Filardo et al., 2000, 2002; Filardo, 2002). Interestingly, the selective estrogen receptor modulator (SERM) Tamoxifen and antiestrogen ICI 182,780 both promote GPR30-dependent transactivation of the EGF receptor and subsequent MAPK activation. Therefore, the pharmacology of GPR30 as defined in cancer cells is different from that of Gq-mER in neurons (Lagrange et al., 1997; Qiu et al., 2003, 2006b), suggesting that these are different receptors. In fact, the mER-mediated response to estrogen in arcuate neurons is still present in GPR30 KO mice (Qiu et al., 2008). Although GPR30 has been localized in the brain and estrogen binds to this receptor (Funakoshi et al., 2006; Bologa et al., 2006; Brailoiu et al., 2007; Prossnitz et al., 2007), further studies of GPR30 are necessary to delineate its function in the brain. It is, however, evident that estrogen can rapidly alter cell function through ERa, ERb, and/or novel ERs. 32.3.3 Membrane-Initiated Signaling of Estrogen It has become clear that there are indirect genomic actions of estrogen in the brain that do not require nuclear targeting of ERs and therefore are classified as membrane-initiated signaling of estrogen (for

review, see Kelly and Levin (2001), Bryant et al. (2006), and Hammes and Levin (2007)). Such signals that are initiated by E2 at the plasma membrane can trigger intracellular signaling events that result in gene transcription. New gene transcription can result from E2 activation of multiple intracellular kinase cascades, including MAPK, phosphoinositide 3-kinase (PI3K), cAMP-protein kinase (PKA), and PKC pathways (Watters et al., 1997; Bi et al., 2001; Cato et al., 2002; Yang et al., 2003; Deisseroth et al., 2003). These events are often termed rapid signaling cascades because they are initiated at the plasma membrane or in the cytoplasm and occur much faster than ERE-driven events (Bryant et al., 2005; Bjo¨rnstro¨m and Sjo¨berg, 2005). For example, E2 binding to its known receptors or a novel mER can upregulate cAMP in hypothalamic neurons by increasing adenylyl cyclase activity (Lagrange et al., 1997). cAMP activates PKA, which in turn phosphorylates CREB and elicits new gene transcription (Zhou et al., 1996; Gu et al., 1996; Watters and Dorsa, 1998; Abraham et al., 2004). Therefore, genes with CRE-binding sites can be activated within a relatively short time course in neurons independent of ERs interacting with EREs. These actions of estrogen via phosporylation of CREB (pCREB) result in activation of genes encoding neurotransmitters such as dopamine, enkephalin, dynorphin, and neurotensin, which are all critical for hypothalamic function (Gu et al., 1996; Watters and Dorsa, 1998). Moreover, in hypothalamic (mouse) GnRH neurons the rapid phosphorylation of CREB following E2 treatment is observed in ERa but not in ERb KO mice, indicating a role for ERb in the acute activation by pCREB in GnRH neurons (Abraham et al., 2003).

32.4 Functional Consequences of Membrane-Initiated E2 Signaling 32.4.1

17b-Estradiol and Neuroprotection

17b-Estradiol is a known neuroprotective agent in the cortex, striatum, and hippocampus (Singer et al., 1999; Honda et al., 2000; Alkayed et al., 2001; Dubal et al., 2001; Jover et al., 2002; Rau et al., 2003). The neuroprotective effects of estrogen, although less well defined in humans (Bushnell et al., 2006), have been clearly established in animal models (Bryant et al., 2006). The fact that treatment with estradiol in ovariectomized animals dramatically reduces lesions caused by the occlusion of vessels (e.g., middle

Membrane-Initiated Effects of Estrogen in the Central Nervous System

cerebral artery) providing blood supply to these brain regions propelled the field into an extensive pursuit of the potential mechanisms (Dubal et al., 1998; Alkayed et al., 2001; Jover et al., 2002; Gulinello et al., 2006). Interestingly, E2 is neuroprotective when administered acutely or chronically, but only at the time of OVX and not after a long period (10 weeks) of estrogen deficiency, which may help explain some of the controversies concerning estrogen and neuroprotection in postmenopausal women (Bushnell et al., 2006; Wassertheil-Smoller et al., 2003; Suzuki et al., 2007). There is substantial evidence that estrogen is neuroprotective via membrane-initiated signaling mechanisms such as the MAPK and PI3K pathways, as well as through modulation of anti-apoptotic Bcl-2 proteins (Mendez et al., 2005; Bryant et al., 2006; Mannella and Brinton, 2006; Jover-Mengual et al., 2007; Zhao and Brinton, 2007). It appears that E2 activation of PI3K is required for subsequent downstream activation of pAkt and pERK1 and -2 (Mannella and Brinton 2006). ERa and perhaps ERb are involved since neuroprotection is lost (aERKO) or is attenuated (bERKO) in animals that lack the respective receptors (Dubal et al., 2001, 2006; Zhao et al., 2004). Thus, it has been proposed that E2 activation of PI3K is mediated by an interaction between ERs and the regulatory protein of PI3K, p85, in cortical neurons (Mannella and Brinton, 2006). According to this model, ER interacts with the regulatory p85 subunit of PI3K, which facilitates the translocation of the catalytic p110 domain to the membrane and subsequent interaction with PI3K substrates (Mannella and Brinton, 2006). Importantly, the ER/ p85 protein interaction is a critical and necessary step required for PI3K-dependent activation of ERK and Akt, and subsequent neuroprotection. In hippocampal CA1 neurons, estradiol acts via ERs and IGF-I receptors to induce ERK/MAPK signaling and subsequent CREB activation to promote neuronal survival following ischemia ( Jover-Mengual et al., 2007). The specific mechanism of interaction between ERs and IGF-I receptors in hippocampal neurons is currently unknown. In COS cells stimulation with E2 promotes binding of ERa, but not ERb, to the IGF-I receptor (Kahlert et al., 2000). However, prior phosphorylation of ERK1,-2 is required for this binding to occur. Therefore, further studies are needed to elucidate the mechanism by which estradiol initiates the signaling necessary for neuroprotection. An interesting model for E2-mediated neuroprotection is that estrogen induces phosphorylation of signal transducer and

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activator of transcription-3 (pSTAT-3) in response to cerebral ischemia (Dziennis et al., 2007). STAT proteins are transcription factors that reside in the cytoplasm in an un-phosphorylated state (Bromberg, 2001; Levy and Darnell, Jr., 2002). When these proteins are activated by phosphorylation in response to cell-surface stimuli, such as cytokines, growth factors, and hormones, they dimerize and translocate to the nucleus to induce gene transcription (Levy and Darnell, Jr., 2002; Reich and Liu, 2006). STAT-3 is phosphorylated in the cytoplasm of cortical cells and transported to the nucleus within 3h after middle cerebral artery occlusion and estradiol treatment enhances STAT-3 phosphorylation (Dziennis et al., 2007). The activation of STAT-3 occurs primarily in neurons of the peri-ischemic cortex as revealed with immunoreactive co-localization of phosphorylated STAT-3 and the neuronal marker MAP2 (Figure 2). Importantly, the inhibition of STAT-3 phosphorylation with JSI-124 abolishes the ability of estradiol to protect against ischemic brain injury, suggesting that estradiol uses STAT-3 as a mediator of neuroprotection. The specific mechanism by which estradiol phosphorylates STAT-3 is currently unknown. 32.4.2 17b-Estradiol and Nigrostriatal Motor Pathways Clinically, the corpus striatum of the basal ganglia is probably one of the most important target areas for estrogen’s actions outside the hypothalamus. The corpus striatum consists of the caudate and putamen, both of which receive projections from the substantia nigra dopamine neurons. Tardive dyskinesia, a disorder that is characterized by involuntary choreatic or dystonic movements which develops after prolonged antipsychotic drug (e.g., haloperidol) exposure, has a 2:1higher incidence in women versus men (Tepper and Haas, 1979; Kompoliti, 1999). In addition, postmenopausal estrogen replacement therapy is associated with a reduced risk of Parkinson’s disease in women and a lower disease severity in women with early Parkinson’s disease who are not taking L-DOPA (Saunders-Pullman et al., 1999). Although the striatum is a prime target for estrogen modulation of motor activity (Kompoliti, 1999), there is virtually no ERa mRNA expression and very little ERb mRNA expression (Simerly et al., 1990; Shughrue and Merchenthaler, 2001; Mitra et al., 2003) in this dopamine-rich pathway suggesting that E2 activates another receptor subtype in this region.

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(c) Figure 2 Co-localization of pSTAT-3, MAP2, and Bcl-2 expression in the peri-ischemic cerebral cortex after middle cerebral artery occlusion (MCAO) in estradiol-replaced OVX rats. (a) Schematic drawing of a coronal rat brain slice illustrating the areas used for cortical dissections and immunohistochemical analysis (shaded area). The same designated areas were used for the contralateral side. (b) Photomicrographs from mid-ischemic (striatum) and peri-ischemic (cortex) regions on the ipsilateral and contralateral sides. The neuronal marker MAP2 (green) was highly expressed in most brain regions, including the peri-ischemic cortex and the contralateral cortex, but not in mid-ischemic tissue. pSTAT-3 (red) was densely expressed in the peri-ischemic area only, which co-localized with MAP2. (c) pSTAT-3 (red) and Bcl-2 (green) exhibited a similar distribution pattern and co-localized in most peri-ischemic cortical cells. Insets illustrate higher-power examples of the immunoreactivity. Images are representative of four immunohistochemical runs from four different animals. Scale bars, 100 mm. Reproduced from Dziennis S, Jia T, Rønnekleiv OK, Hurn PD, and Alkayed NJ. Role of signal transducer and activator of transcription-3 in estradiol-mediated neuroprotection Journal of Neuroscience 27: 7268–7274, 2007, with permission from the Society for Neuroscience. Copyright 2007 by the Society for Neuroscience.

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Estrogen has multiple short-term effects in the corpus striatum. It potentiates rotational behavior induced by amphetamine in ovariectomized, 6-hydroxydopamine-(unilateral) lesioned rats (Becker, 1990). Besides this pronounced behavioral effect on amphetamine-induced rotational behavior, acute administration of E2 also increases amphetamineinduced striatal dopamine release within 30min as measured by in vivo microdialysis (Becker, 1990), increases striatal dopamine turnover (Di Paolo et al., 1985), and uncouples and downregulates dopamine D2-receptors within the same time period (Levesque and Di Paolo, 1988; Bazzett and Becker, 1994). This is consistent with an earlier report that fewer caudate neurons are inhibited by dopamine, presumably due to D2 dopamine receptor downregulation, following acute E2 treatment (Arnauld et al., 1981). A similar effect of E2 has been shown in the nucleus accumbens, a target site of ventral tegmental dopamine neurons, in which local injections of E2 (20–40pg) and not 17a-estradiol rapidly (within 2min) potentiate Kþ-stimulated dopamine release as measured by in vivo voltametry (Thompson and Moss, 1994). Interestingly, continued exposure to E2 (2weeks) downregulates D2 receptor mRNA (Lammers et al., 1999), which fits with E2 downregulation of other Gai,o-coupled receptors throughout the nervous system (for review, see Kelly and Wagner (1999)). Longterm exposure to E2 also potentiates D1-stimulated adenylyl cyclase activity and attenuates D2-inhibition of adenylyl cyclase in striatal neurons (Maus et al., 1989), a differential effect on Gas-coupled receptors versus Gai,o-coupled receptors. Furthermore, E2 enhances the pertussis toxin-catalyzed ADP-ribosylation of Gai,o (Maus et al., 1990), which indicates that E2 modifies the G-protein, possibly through PKA phosphorylation. This is very analogous to the rapid effects of E2 in the hypothalamus (Lagrange et al., 1997) and in the hippocampus (Gu and Moss, 1996, 1998); therefore, this mechanism of uncoupling Gai,o-coupled monoamine receptors may be via a common pathway in the CNS and peripheral tissues (Kelly and Wagner, 1999). The effect of E2 on presynaptic dopamine D2 receptor coupling in the striatum would attenuate the ability of dopamine to inhibit dopamine release and hence result in an increase in dopamine turnover in the striatum (Di Paolo et al., 1985; Becker, 1990). Evidence for a membrane receptor for E2 is further substantiated by studies showing that membrane impermeable E2-BSA rapidly stimulates dopamine release from

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striatal slices (Ramirez and Zheng, 1996). At the cellular level, E2 and E2-BSA rapidly inhibit whole-cell L-type calcium currents (characterized with selective L-type calcium channel blockers) in medium spiny (GABAergic) caudate-putamen neurons with femtomolar potency (Mermelstein et al., 1996). The effects of E2 are dependent on the sex in that striatal neurons from females exhibit about twofold greater inhibition than neurons from males. The inhibition is rapidly reversible upon washing out of the steroid, but whole-cell dialysis with GTPgS, which inhibits the G-protein recycling, blocks the recovery from E2 inhibition. Interestingly, the reduction in the peak Ca2þ current by E2 diminishes with repeated application which indicates that there is a desensitization of the ER-signaling pathway and/or a rundown of the estrogen-sensitive current. A property of GPCRs is that they desensitize and downregulate with continued exposure to agonist, which lends further credence to the notion that E2 is interacting with a G-protein-coupled membrane receptor to activate intracellular cascades. 32.4.3 17b-Estradiol, Growth Factors, and Reproduction Systemic administration of E2 in ovariectomized rats activates IGF-1 receptors and induces the association between IGF-1 receptors and ERa in the hypothalamus (Quesada and Etgen, 2001; Cardona-Go´mez et al., 2002; Mendez et al., 2003). Similar to the effects in cortical neurons, there is an interaction (complex formation) between the p85 subunit of PI3K and ERa within 1–3h, which leads to activation of Akt (Cardona-Go´mez et al., 2002; Mendez et al., 2003). Also, the E2-induced activation of IGF-1 receptors augments a1-adrenergic receptor signaling, which is important for reproductive functions (Quesada and Etgen, 2001). On the other hand, blockade of IGF-1 receptors during E2 priming prevents E2-induced increases in a1-adrenergic receptor-binding density as well as IGF-1 enhancement of noradrenergic receptor signaling (Quesada and Etgen, 2002). Collectively, these findings support functional interactions between E2 and IGF-1. Therefore, these actions of E2 on the IGF-1 receptor signaling pathway may be a key mechanism by which estrogen affects synaptic remodeling and neuronal plasticity during the estrous cycle. Of particular interest are the findings that intracerebroventricular (ICV) infusion of JB-1, a selective competitive antagonist of IGF-1 autophosphorylation,

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inhibits the estrogen-induced LH surge and sexual behavior in ovariectomized rats (Quesada and Etgen, 2002). In addition, co-administration (ICV) of blockers of PI3 kinase (wortmannin) and MAPK (PD98059) inhibit the long-term (48h) effects of E2 to induce the LH surge and facilitate lordosis behavior (Etgen and Acosta-Martinez, 2003). Therefore, facilitation of female sexual behavior by E2 appears to involve activation of both PI3 kinase and MAPK signal transduction pathways. The importance of growth factors for female sexual behavior is further illustrated by observations that EGF and also IGF-1 can, in the absence of estrogen and progesterone and within 1–4 h of ICV administration, induce mating behavior in rats and mice, in part, through an ERa-dependent mechanism (Apostolakis et al., 2000). This relatively rapid, ligand-independent ER action is in striking contrast to the well-established finding that estrogen priming over a period of at least 24 h is needed for progesterone induction of female reproductive behavior (Etgen et al., 2001). The crosstalk between estrogen signaling and membrane-initiated growth factor signaling in the hypothalamus is particularly interesting, although it is currently not well understood. The ability of both IGF-1 and estradiol to induce female sexual behavior may involve complex interactions between ERa, the IGF-1 receptor, and the PI-3 kinase p85 subunit. Further discussions on this topic can be found in Chapter 33, Estrogen Regulation of Neurotransmitter and Growth Factor Signaling in the Brain. 32.4.4 17b-Estradiol and GnRH Neurosecretion Despite having been studied extensively for over 25 years, the mechanism(s) by which estrogen regulates GnRH neurons is not well understood. It has been obvious for a number of years that GnRH neurons are modulated by estrogen in a complex manner. For example, loss of estrogen by ovariectomy disrupts GnRH regulation of pituitary LH secretion and results in elevated levels of plasma LH. This effect is due to the loss of negative feedback actions of E2. However, both negative and positive (induction of the LH surge) feedback regulation of LH (GnRH) secretion can be restored by replacement with E2. The positive feedback is believed to be by an action of E2 in the anteroventral periventricular (AVPV) nucleus (Herbison 1998; Han et al., 2005; Smith et al., 2006). Neurons in the AVPV express kisspeptin (a neuropeptide encoded by the kiss gene) and GABA, both

of which are important for regulation of GnRH neurosecretion (Wagner et al., 2001a; Jackson and Kuehl, 2002; DeFazio et al., 2002; Smith et al., 2006; Christian and Moenter, 2007). The AVPV area expresses high levels of ERa and also ERb, and the actions of the gonadal steroids are mediated, in part, via the nuclear-initiated signaling (genomic) mechanism (Shughrue et al., 1997; Wintermantel et al., 2006). However, the AVPV is also sensitive to the rapid actions of gonadal steroids. For example, E2 within 30 min increases the expression of the pCREB in the AVPV (Gu et al., 1996). At the cellular level, individual AVPV neurons including GABA neurons respond to both b-adrenergic and a-adrenergic input by a reduction in the median after-hyperpolarization current (mIAHP), which increases the action potential firing in these neurons (Wagner et al., 2001b). Moreover, the a1-adrenergic, but not b-adrenergic inhibition of the mIAHP is potentiated after acute (15–20min) exposure to estrogen, which further increases neuronal excitability (Wagner et al., 2001b). The estrogeninduced enhancement of the coupling of the a1-adrenergic receptors to their effector calcium-activated Kþ (SK) channels (underlying the mIAHP) is initiated within 15min in vitro and lasts for at least 24 h following systemic steroid administration, suggesting both rapid and sustained effects (Wagner et al., 2001b). Since SK channels are critical for modulating neuronal firing rate and pattern (Stocker et al., 1999; Sah and Davies, 2000), estrogen-induced modulation of these channels would have significant functional consequences for AVPV neurons and their targets. Because ERa, the first cloned receptor/transcription factor for E2, has not been localized to native GnRH neurons, the predominant view has been that estrogen affects GnRH neurons through presynaptic mechanisms. However, in the presence of tetrodotoxin (TTX) which blocks fast Naþ channel activity and thus prevents synaptic inputs, GnRH neurons are rapidly hyperpolarized by estrogen, an effect that inhibits the activity of these neurons (Kelly et al., 1984; Condon et al., 1989; Lagrange et al., 1995). These findings, therefore, suggest a direct hyperpolarizing action of estrogen on GnRH neurons via a Gai,o-coupled receptor. Indeed, in GT1–7 cells, an immortalized GnRH neuronal cell line, estrogen inhibits adenylyl cyclase activity (cAMP production) via a pertussis toxin (Gai,o-coupling) mechanism (Navarro et al., 2003). The electrophysiological effects are much too rapid to involve transcription through classical ERs, but an estrogen–Gai,o coupled receptor has not been identified. Interestingly, E2 increases the firing in

Membrane-Initiated Effects of Estrogen in the Central Nervous System

primate nasal placode GnRH neuronal cultures within 10 min (Abe and Terasawa, 2005). Therefore, E2 may have both direct inhibitory and indirect (?) excitatory effects on GnRH neurons, since the latter experiments were done in the absence of blockade of synaptic input. An important milestone for understanding estrogen’s action in GnRH neurons was the discovery of a second ER, ERb, in 1996 and the documentation that this receptor was expressed in GnRH neurons (Kuiper et al., 1996; Hrabovszky et al., 2000, 2001; Kallo et al., 2001; Herbison and Pape, 2001). The latter findings combined with recent technological advances, such as the development of ER mutants and transgenic animals expressing green fluorescent protein (GFP) in GnRH neurons, have greatly facilitated studies to understand the cellular mechanisms by which GnRH neurons are regulated by estrogen (Spergel et al., 1999; Suter et al., 2000; Kato et al., 2003; Han et al., 2005; Abraham et al., 2003; Smith et al., 2006; Wintermantel et al., 2006; Zhang et al., 2007). A series of recent publications are particularly enlightening because they show that in mouse hypothalamic neuronal explants and in primate nasal placode cultures, E2 augments synchronous intracellular Ca2þ oscillations in GnRH neurons (Temple et al., 2004; Temple and Wray, 2005; Abe et al., 2008). This relatively rapid effect is observed within 10–30min of E2 application. The calcium oscillations in the presence of TTX are most likely the result of estrogen action directly on GnRH neurons. Furthermore, E2 conjugated to BSA at the C-17 position (E2-17 BSA) or to a large nondegradable poly(amido) amine-dendrimer macromolecule (Harrington et al., 2006) mimicked the effects of E2 on intracellular calcium oscillations (Temple et al., 2004; Temple and Wray, 2005; Abe et al., 2008). However in the mouse explant experiments (Temple et al., 2004; Temple and Wray, 2005), the E2-induced calcium oscillations were blocked by ICI 182,780suggesting that an ER is involved. In contrast, E2-induced calcium oscillations in the primate placode cultures were not blocked by ICI 182,780, and the antagonist ICI 182,780 alone had no effect suggesting that an ER may not be involved (Abe et al., 2008). The actions of E2-BSA in mouse GnRH neurons were abrogated by pertussis toxin treatment and not by inhibition of gene transcription, supporting a role for a G protein-coupled membrane receptor (Temple et al., 2004; Temple and Wray, 2005). However, in vivo studies have provided evidence for ERb in the E2-induced rapid (15min) induction of pCREB in mouse GnRH neurons

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(Abraham et al., 2003). Collectively, these findings indicate that there are multiple acute actions of E2 in GnRH neurons, some of which involve ERb. Interestingly, mice lacking ERb appear to exhibit normal sexual behavior and reproduce successfully with the exception that their litter size is reduced, presumably due to a decline in ovarian function (Krege et al., 1998; Dupont et al., 2000). In contrast, ERa, which is not expressed in GnRH neurons, is essential for both sexual behavior and fertility (Ogawa et al., 1998; Dupont et al., 2000). Therefore, the functional significance of ERb in GnRH neurons remains uncertain. In fact, how the rapid actions of the steroids are related to the longer-term genomic actions are currently not well understood. For example, recent findings have revealed that GnRH neurons express K-ATP channels and the agonist-induced outward (K-ATP channel) current is increased by approximately. twofold in E2-treated animals (Zhang et al., 2007). The increased activity, which occurs in the presence of TTX and GABA and glutamate blockade, does not appear to be the result of increased expression of the channel based on quantitative real-time PCR results. Therefore, it appears that there is an E2induced change in signaling molecules (kinases) that impinge on the channel, part of which may be nuclearinitiated steroid signaling and/or acute phosphorylation events. Clearly, further studies are needed to elucidate the regulation of GnRH neurons by E2. Studies are forthcoming on the crosstalk between rapid membrane-initiated and long-term nuclearinitiated steroid actions (Lagrange et al., 1994, 1997; Wagner et al., 2001b; Vasudevan et al., 2001; Kow and Pfaff, 2004; Malyala et al., 2005; Qiu et al., 2006a,b; Roepke et al., 2007). For example, it has been found that both acute effects of E2 and the transcriptional changes alter excitability of hypothalamic neurons (Kelly et al., 2003; Malyala et al., 2005; Qiu et al., 2006a). In addition, the estrogen-induced membrane actions in the VMH can potentiate its genomic effects on lordosis behavior (Kow and Pfaff 2004). The E2-induced membrane effects can perhaps be attributed to its potentiation of Naþ currents and attenuation of Kþ currents in VMH neurons (Kow et al., 2006). Moreover, the membrane effects in the VMH appear to be at least in part mediated by signaling pathways involving PKC and PKA (Kow and Pfaff, 2004). These findings are particularly intriguing in view of other findings that estrogen activates a novel mER, other than ERa or ERb that is Gaq-coupled to PLC, PKCd, and PKA to alter cell firing (Qiu et al., 2003, 2006b).

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32.4.5 Effects of 17b-Estradiol on VMH/ Arcuate Neurons: Role in Regulation of Feeding In addition to its role in the control of reproduction, estrogen is involved in the regulation of appetite, energy expenditure, body weight, and adipose tissue deposition and distribution in females (Milewicz et al., 2000; Geary, 2001; Poehlman, 2002). Ovariectomy induces an increase in food intake and decreases ambulatory and wheel-running activities in rodents, all of which are reversed with estrogen replacement (Ahdieh and Wade, 1982; Colvin and Sawyer, 1969; Shimomura et al., 1990; Asarian and Geary, 2002). In fact, hypo-estrogenic states are associated with decreased activity and an increase in body weight in females (Czaja and Goy, 1975; Butera and Czaja, 1984; Czaja, 1984; McCaffrey and Czaja, 1989; Jones et al., 2000; Asarian and Geary, 2002; Qiu et al., 2006b; Clegg et al., 2006, 2007). The anorectic effects of estrogen are thought to be mediated through CNS actions based on the findings that direct injections of E2 into the PVH or arcuate/VMH are effective to reduce food intake, body weight, and increase wheel-running activity in females (Colvin and Sawyer, 1969; Ahdieh and Wade, 1982; Butera and Czaja, 1984). Also, specific repression of ERa in the VMH leads to obesity, hyperphagia, and reduced energy expenditure (Musatov et al., 2007). Therefore, it is evident that neurons in these hypothalamic nuclei regulate energy homeostasis and are affected by E2. In the arcuate nucleus, E2 upregulates the expression of the peptide b-endorphin in POMC neurons in ovariectomized female guinea pigs (Thornton et al., 1994; Bethea et al., 1995). Furthermore, there is a decrease in hypothalamic b-endorphin levels in the hypothalamus of postmenopausal women who do not take hormone replacement that correlates with weight gain (Leal et al., 1998). In contrast, E2 reverses the ovariectomy-induced increase in arcuate NPY mRNA expression in rodents (Shimizu et al., 1996). Therefore, it appears that the arcuate nucleus and specifically POMC neurons are a major target for the anorectic actions of estrogen, which underscores their importance in the control of energy homeostasis. Indeed, POMC neurons are critical for the regulation of feeding and are also involved in the rewarding aspects of food intake (Hayward et al., 2002; Appleyard et al., 2003). Important inhibitory regulators of POMC neuronal activity are the G-protein-activated, inwardly rectifying potassium (GIRK) channels. Both m-opioid

receptor (e.g., by b-endorphin or by selective m-opioid agonists) or GABAB receptor activation of these GIRK channels directly hyperpolarize and thereby inhibit hypothalamic neurons (Loose et al., 1990; Kelly et al., 1992; Lagrange et al., 1994, 1995, 1996). Brief (<20min) application of E2 or BSA-E2 in vitro causes a fourfold decrease in the potency of m-opioid receptor agonists and GABAB receptor agonists to inhibit POMC neurons (Lagrange et al., 1994, 1996; Qiu et al., 2003), indicating that an mER is involved. As stated previously, this mER is distinct from ERa or ERb (Qiu et al., 2003, 2006b). Therefore, E2 via an mER-mediated pathway rapidly decreases the potency of m-opioid and GABAB ligands at their receptors (desensitization), thus increasing POMC neuronal firing and the release of the POMC products, b-endorphin and melanocyte-stimulating hormone (MSH). Interestingly, inwardly rectifying Kþ channels are also a point of convergence for the effects of leptin and insulin in POMC neurons (Plum et al., 2006). Both leptin and insulin activate PI3K (via insulin receptor substrate) that leads to the metabolism of phosphatidylinositol 4,5-biphosphate (PIP2) to phoshatidylinositol 3,4,5-triphosphate (PIP3) in POMC neurons (Xu et al., 2005). Activation of GIRK channels requires permissive levels of membrane PIP2 and increased channel activity results from Gbgmediated stabilization of PIP2-GIRK binding (Huang et al., 1998; Zhang et al., 1999). In fact, the regulation of channel activities by Gbg, PIP2, or phosphorylation occurs on the timescale of a few seconds which can be resolved by electrophysiological techniques (Suh and Hille, 2002). In recent studies from our lab we have found that PI3K plays a critical role in facilitating the rapid membrane response to estrogen, as elucidated by the use of PI3K inhibitors (Malyala et al., 2008). Indeed, we found that PI3K inhibitors, which block the phosphorylation of PIP2 and thereby increase the levels of PIP2 in the membrane, augmented GIRK channel activity and attenuated the effects of E2 on the mER signaling pathway. As proof of principle of the importance of the membrane-initiated estrogen signaling pathway in the control of energy homeostasis, we used STX to selectively target the mER (i.e., in vivo treatment), and found that STX, similar to E2, attenuated the weight gain following ovariectomy (Figure 3) (Qiu et al., 2006b). Moreover, the fact that both E2 and STX are fully efficacious in activating this signaling pathway in double-ER KO mice (Figure 4) is

Membrane-Initiated Effects of Estrogen in the Central Nervous System

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Figure 3 Estradiol (a) and STX (b) significantly attenuate the body weight gain in female guinea pigs after ovariectomy. The female guinea pigs were ovariectomized on day 0 and allowed to recover for 1week before being given bi-daily subcutaneous injections of oil (OIL), estradiol benzoate (EB), or STX. A two-way ANOVA (repeated measures) revealed an overall significant effect of both EB and STX ( p < 0.001), and post hoc Newman–Keuls analysis revealed daily significant differences between EB and oil-treated, and STX and oil-treated groups (**p < 0.01) (c) Uteri are enlarged after estradiol, but not after STX or oilvehicle treatment (inset). After the treatment period, the uteri of the guinea pigs were harvested and examined. There was a significant increase in uterine size after EB, as compared with oil-vehicle or STX treatment. Bars represent the mean SEM of six and four animals per group for EB and STX treatment, respectively. ***p < 0.001, EB vs. oil-treated

The gonadal steroid estrogen participates in numerous functions including reproduction, feeding, neuroprotection, and cognition. It has been known for sometime that the main actions of gonadal steroids are to regulate gene transcription through binding to and activating nuclear receptors that can stimulate or inhibit gene transcription at specific DNA-binding sites. However, recently it has become clear that these gonadal steroids exert their action through multiple signaling mechanisms, including membraneinitiated, cytoplasmic, as well as nuclear-initiated steroid signaling. For example, there is evidence that estrogen increases the activity of hypothalamic POMC and dopamine neurons through binding to a membrane-localized receptor distinct from ERa or ERb (Qiu et al., 2003, 2006b). In addition, estrogen conjugated to macromolecules can activate calcium pulses in GnRH neurons by a mechanism that appears to be independent of ERb (Temple and Wray, 2005; Abe et al., 2008). There are many examples in the hypothalamus and other brain regions for an E2-induced upregulation of PKC, PKA, PI3K, and MAPK activity leading to altered neuronal activity, as well as altered behavioral activities (Figure 5). Although the exact identity of the membraneassociated ER is still not known, we are making substantial progress toward a better understanding of the full complement of steroid hormone actions in the brain. Also, we are beginning to understand the interaction between rapid signaling events and changes in gene expression. However, many challenges lie ahead to fully identify the nature of membrane steroid receptors, functional properties, and integration (crosstalk) with nuclear steroid receptors.

females. Reproduced from Qiu J, Bosch MA, Tobias SC, et al. (2006) A G-protein-coupled estrogen receptor is involved in hypothalamic control of energy homeostasis. Journal of Neuroscience 26: 5649–5655, with permission from the Society for Neuroscience. Copyright 2006 by the Society for Neuroscience.

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Figure 4 E2 and STX rapidly attenuated the baclofen response in hypothalamic arcuate (POMC) neurons in wildtype and ERa, ERb, and ERab knockout (ERaKO, ERbKO, and ERabKO) mice. (a) Four representative traces of the baclofen response in the presence of vehicle, E2 (100nM) or STX (100nM) in arcuate neurons from wildtype (A and B) and ERaKO (C and D) mice are shown. (b) Bar graphs summarizing the effects of E2 or STX in arcuate (POMC) neurons from ovariectomized, wildtype, ERaKO, and ERbKO mice. E2 and STX (100nM) rapidly attenuated the outward (GIRK) current induced by the GABAB receptor agonist baclofen in arcuate (POMC) neurons from ovariectomized wildtype (C57BL/6), ERaKO, and ERbKO mice. Bars represent the mean SEM. ###p < 0.001, E2 treated wildtype group vs. vehicle wildtype control; ***p < 0.001, E2 or STX treated ERaKO or ERbKO groups vs. vehicle ERKO control. The data for the ERaKO and ERbKO control responses were combined in one bar graph. (c) Double labeling of POMC neurons that responded to STX from ERaKO (A–C) and ERbKO (D–F) mice. Arcuate neurons were filled with biocytin during the whole-cell recording. A, D, Biocytin-streptavidin-Cy2 labeling of two small pyramidal arcuate neurons. B, E, Immunocytochemical staining of bendorphin in the same neurons (arrow). C, overlay of A and B; F, overlay of D and E. Nineteen of the 46 responsive neurons (41%) were identified as b-endorphin neurons. Scale bar¼20 mm in A–F. (d) Bar graphs summarizing the effects of E2, STX or ICI 182,780 in arcuate (POMC) neurons from male ERabKO mice. E2 and STX (100nM) rapidly attenuated the outward (GIRK) current induced by the GABAB receptor agonist baclofen, which was antagonized by ICI. ICI alone had no effect. Twelve of 20 (60%) were identified as POMC neurons. Bars represent the mean SEM {{{p < 0.001, E2 or STX vs. vehicletreated cells from ERabKO mice. Reproduced from Qiu et al., A G-protein-coupled estrogen receptor is involved in hypothalamic control of energy homeostasis. Journal of Neuroscience 26: 5649–5655, 2006 permission from the Society for Neuroscience. Copyright 2006 by the Society for Neuroscience.

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Figure 5 Schematic overview representing the estrogen-mediated modulation of G-protein-coupled receptors via a membrane-associated receptor (mER) in hypothalamic neurons. E2 binds to an mER that is Gaq-coupled to activate phospholipase C 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 protein kinase C (PKC). Through phosphorylation, adenylyl cyclase (AC) activity is upregulated by PKC. The generation of cAMP activates PKA, which can uncouple m-opioid (m) and GABAB receptors from their signaling pathway through phosphorylation of a downstream effector molecule (e.g., the inwardly rectifying Kþ channel, or GIRK). PKA can potentially phosphorylate other channels (e.g., K-ATP channels) to alter their function. The b1-adrenergic (b1) receptor is Gas-coupled to activation of an adenylyl cyclase-PKA pathway that inhibits the small conductance, Ca2þ-activated Kþ channel (SK). The a1-adrenergic receptor is Gaq-coupled to the phosphatidylinositol pathway to activate PKC and PKA to phosporylate the SK channel and inhibit its activity, which is potentiated by E2. ER-mediated modulation of kinase pathways either reduces the capacity of neurotransmitters such as b-endorphin (bEnd) and GABA to inhibit hypothalamic neuronal excitability or augments the ability of neurotransmitters such as norepinephrine (NE) to increase neuronal excitability. The membrane-initiated steroid (E2) signaling through PKA can phosphorylate cAMP-responsive element binding protein (pCREB), which can alter gene transcription through its interaction with the cAMP responsive element (CRE). Therefore, the rapid membrane-initiated signaling can alter gene expression in an estrogen-response element-independent fashion.

Acknowledgments

References

The authors thank members of their laboratories who contributed to the work described herein, especially Drs. Jian Qiu, Chunguang Zhang, Troy A. Roepke, Anna Malyala, and Ms. Martha A. Bosch; also, special thanks to Ms. Martha A. Bosch for her skilled assistance with the illustrations and manuscript preparation. Dr. Troy A. Roepke provided helpful comments on earlier versions of the manuscript. The work from the authors laboratories was supported by PHS grants NS 43330, NS 38809, DK 68098, and P01 NS049210.

Abe H, Keen KL, and Terasawa E (2008) Rapid action of estrogens on intracellular calcium oscillations in primate LHRH-1 neurons. Endocrinology 149: 1155–1162. Abe H and Terasawa E (2005) Firing pattern and rapid modulation of activity by estrogen in primate luteinizing hormone releasing hormone-1 neurons. Endocrinology 146: 4312–4320. Abraham IM, Han SK, Todman MG, Korach KS, and Herbison AE (2003) Estrogen receptor beta mediates rapid estrogen actions on gonadotropin-releasing hormone neurons in vivo. Journal of Neuroscience 23: 5771–5777. Abraham IM, Todman MG, Korach KS, and Herbison AE (2004) Critical in vivo roles for classical estrogen receptors in rapid

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Biographical Sketch

Dr. Rønnekleiv is a professor in the Department of Physiology and Pharmacology, professor in the Department of Anesthesiology and Perioperative Medicine, Oregon Health and Science University (OHSU), and a senior scientist in the Division of Neuroscience, Oregon National Primate Research Center (ONPRC). She earned her PhD in physiology under the guidance of Dr. SM McCann from the University of Texas Southwestern Medical School in 1974. After postdoctoral research at the Max Planck Institute for Biophysical Chemistry in Go¨ttingen, Germany, as an Alexander von Humboldt fellow, Dr. Rønnekleiv joined the Department of Psychiatry, University of Pittsburgh School of Medicine as an assistant professor in 1979. She moved to Portland, Oregon in 1982 with joint appointments at OHSU and ONPRC. Dr. Rønnekleiv was promoted to associate professor in 1987 and to professor in 1998. Dr. Rønnekleiv has been continually funded from NIH since 1981, and her research has focused on the actions of estrogen in the brain to control motivated behavior such as eating, drinking, and reproduction. She discovered in primates that GnRH neurons originate in the nose, and has had a particular interest in the mechanism by which these neurons are regulated by estrogen. Recently, Dr. Rønnekleiv, as part of a Program Project Grant on Sex Steroids and Neuroprotection, has embarked on studies to understand the mechanism by which estrogen protects neurons.

Martin J. Kelly received his PhD in physiology in 1976 from the University of Texas Southwestern Medical School at Dallas. He did his graduate work on the rapid effects of estrogen on hypothalamic neuronal activity under the guidance of Dr. Robert L. Moss (1940–99). He spent 3 years (1976–79) as a postdoctoral fellow in Germany in the Department of Neurobiology at the Max Planck Institute for Biophysical Chemistry. There he developed the hypothalamic slice preparation, which laid the ground work for doing voltage-clamp studies of small neurons and studying ion channels associated with rapid estrogen signaling. In 1979, Dr. Kelly moved to the University of Pittsburgh where he joined the Department of Physiology as an assistant professor. There he received his first NIH funding (R01) to continue the slice

work investigating the membrane-initiated signaling of estrogen. He moved to Oregon Health and Science University in 1981 to continue his work and was a founding member of the neurosciences graduate program at OHSU. Dr. Kelly was promoted to associate professor in 1987 and full professor in 1992. During this period, Dr. Kelly has received two NIH Research Career Development Awards to work on novel estrogen signaling in the brain.