Paul E. Micevych

Direct regulation of adult brain function by the male-specific factor SRY.

The central dogma of mammalian brain sexual differentiation has contended that sex steroids of gonadal origin organize the neural circuits of the developing brain. Recent evidence has begun to challenge this idea and has suggested that, independent of the masculinizing effects of gonadal secretions, XY and XX brain cells have different patterns of gene expression that influence their differentiation and function. We have previously shown that specific differences in gene expression exist between male and female developing brains and that these differences precede the influences of gonadal hormones. Here we demonstrate that the Y chromosome-linked, male-determining gene Sry is specifically expressed in the substantia nigra of the adult male rodent in tyrosine hydroxylase-expressing neurons. Furthermore, using antisense oligodeoxynucleotides, we show that Sry downregulation in the substantia nigra causes a statistically significant decrease in tyrosine hydroxylase expression with no overall effect on neuronal numbers and that this decrease leads to motor deficits in male rats. Our studies suggest that Sry directly affects the biochemical properties of the dopaminergic neurons of the nigrostriatal system and the specific motor behaviors they control. These results demonstrate a direct male-specific effect on the brain by a gene encoded only in the male genome, without any mediation by gonadal hormones.

Membrane Estrogen Receptor-α Interactions with Metabotropic Glutamate Receptor 1a Modulate Female Sexual Receptivity in Rats

In rats, female sexual behavior is regulated by a well defined limbic–hypothalamic circuit that integrates sensory and hormonal information. Estradiol activation of this circuit results in μ-opioid receptor (MOR) internalization in the medial preoptic nucleus, an important step for full expression of sexual receptivity. Estradiol acts through both membrane and intracellular receptors to influence neuronal activity and behavior, yet the mechanism(s) and physiological significance of estradiol-mediated membrane responses in vivo have remained elusive. Recent in vitro evidence found that stimulation of membrane-associated estrogen receptor-α (ERα) led to activation of metabotropic glutamate receptor 1a (mGluR1a). Furthermore, mGluR1a signaling was responsible for the observed downstream effects of estradiol. Here we present data that show that ERα and mGluR1a directly interact to mediate a rapid estradiol-induced activation of MOR in the medial preoptic nucleus, leading to female sexual receptivity. In addition, blockade of mGluR1a in the arcuate nucleus of the hypothalamus resulted in a significant attenuation of estradiol-induced MOR internalization, leading to diminished female sexual behavior. These results link membrane-initiated estradiol actions to neural events modulating behavior, demonstrating the physiological importance of ERα-to-mGluR1a signaling.

Estrogen interacts with the IGF-1 system to protect nigrostriatal dopamine and maintain motoric behavior after 6-hydroxdopamine lesions

The most prominent neurochemical hallmark of Parkinsons disease (PD) is the loss of nigrostriatal dopamine (DA). Animal models of PD have concentrated on depleting DA and therapies have focused on maintaining or restoring DA. Within this context estrogen protects against 6‐hydroxdopamine (6‐OHDA) and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) lesions of the nigrostriatal DA pathway. Present studies tested the hypothesis that neuroprotective estrogen actions involve activation of the insulin‐like growth factor‐1 (IGF‐1) system. Ovariectomized rats were treated with either a single subcutaneous injection of 17β‐estradiol benzoate or centrally or peripherally IGF‐1. All rats were infused unilaterally with 6‐OHDA into the medial forebrain bundle (MFB) to lesion the nigrostriatal DA pathway. Tyrosine hydroxylase (TH) immunocytochemistry confirmed that rats injected with 6‐OHDA had a massive loss of TH immunoreactivity in both the ipsilateral substantia nigra compacta (60% loss) and the striatum (>95% loss) compared to the contralateral side. Loss of TH immunoreactivity was correlated with loss of asymmetric forelimb movements, a behavioral assay for motor deficits. Pretreatment with estrogen or IGF‐1 significantly prevented 6‐OHDA‐induced loss of substantia nigra compacta neurons (20% loss) and TH immunoreactivity in DA fibers in the striatum (<20% loss) and prevented the loss of asymmetric forelimb use. Blockage of IGF‐1 receptors by intracerebroventricular JB‐1, an IGF‐1 receptor antagonist, attenuated both estrogen and IGF‐1 neuroprotection of nigrostriatal DA neurons and motor behavior. These findings suggest that IGF‐1 and estrogen acting through the IGF‐1 system may be critical for neuroprotective effects of estrogen on nigrostriatal DA neurons in this model of PD.

Membrane estradiol signaling in the brain.

While the physiology of membrane-initiated estradiol signaling in the nervous system has remained elusive, a great deal of progress has been made toward understanding the activation of cell signaling. Membrane-initiated estradiol signaling activates G proteins and their downstream cascades, but the identity of membrane receptors and the proximal signaling mechanism(s) have been more difficult to elucidate. Mounting evidence suggests that classical intracellular estrogen receptor-alpha (ERalpha) and ERbeta are trafficked to the membrane to mediate estradiol cell signaling. Moreover, an interaction of membrane ERalpha and ERbeta with metabotropic glutamate receptors has been identified that explains the pleomorphic actions of membrane-initiated estradiol signaling. This review focuses on the mechanism of actions initiated by membrane estradiol receptors and discusses the role of scaffold proteins and signaling cascades involved in the regulation of nociception, sexual receptivity and the synthesis of neuroprogesterone, an important component in the central nervous system signaling.

PI3 Kinase/Akt Activation Mediates Estrogen and IGF-1 Nigral DA Neuronal Neuroprotection Against a Unilateral Rat Model of Parkinson's Disease

Recently, using the medial forebrain bundle (MFB) 6‐hydroxydopmaine (6‐OHDA) lesion rat model of Parkinsons disease (PD), we have demonstrated that blockade of central IGF‐1 receptors (IGF‐1R) attenuated estrogen neuroprotection of substantia nigra pars compacta (SNpc) DA neurons, but exacerbated 6‐OHDA lesions in IGF‐1 only treated rats (Quesada and Micevych [2004]: J Neurosci Res 75:107–116). This suggested that the IGF‐1 system is a central mechanism through which estrogen acts to protect the nigrostriatal DA system. Moreover, these results also suggest that IGF‐1R‐induced intracellular signaling pathways are involved in the estrogen mechanism that promotes neuronal survival. In vitro, two convergent intracellular signaling pathways used by estrogen and IGF‐1, the mitogen‐activated protein kinase (MAPK/ERK), and phosphatidyl‐inositol‐3‐kinase/Akt (PI3K/Akt), have been demonstrated to be neuroprotective. Continuous central infusions of MAPK/ERK and PI3K/Akt inhibitors were used to test the hypothesis that one or both of these signal transduction pathways mediates estrogen and/or IGF‐1 neuroprotection of SNpc DA neurons after a unilateral administration of 6‐OHDA into the MFB of rats. Motor behavior tests and tyrosine hydroxylase immunoreactivity revealed that the inhibitor of the PI3K/Akt pathway (LY294002) blocked the survival effects of both estrogen and IGF‐1, while an inhibitor of the MAPK/ERK signaling (PD98059) was ineffective. Western blot analyses showed that estrogen and IGF‐1 treatments increased PI3K/Akt activation in the SN; however, MAPK/ERK activation was decreased in the SN. Indeed, continuous infusions of inhibitors blocked phosphorylation of PI3K/Akt and MAPK/ERK. These findings indicate that estrogen and IGF‐1‐mediated SNpc DA neuronal protection is dependent on PI3K/Akt signaling, but not on the MAPK/ERK pathway.

Estradiol inhibits atp-induced intracellular calcium concentration increase in dorsal root ganglia neurons.

Estrogen has been implicated in modulation of pain processing. Although this modulation occurs within the CNS, estrogen may also act on primary afferent neurons whose cell bodies are located within the dorsal root ganglia (DRG). Primary cultures of rat DRG neurons were loaded with Fura-2 and tested for ATP-induced changes in intracellular calcium concentration ([Ca(2+)](i)) by fluorescent ratio imaging. ATP, an algesic agent, induces [Ca(2+)](i) changes via activation of purinergic 2X (P2X) type receptors and voltage-gated Ca(2+) channels (VGCC). ATP (10 microM) caused increased [Ca(2+)](i) transients (226.6+/-16.7 nM, n = 42) in 53% of small to medium DRG neurons. A 5-min incubation with 17 beta-estradiol (100 nM) inhibited ATP-induced [Ca(2+)](i) (164+/-14.6 nM, P<0.05) in 85% of the ATP-responsive DRG neurons, whereas the inactive isomer 17 alpha-estradiol had no effect. Both the mixed agonist/antagonist tamoxifen (1 microM) and specific estrogen receptor antagonist ICI 182780 (1 microM) blocked the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. Estradiol coupled to bovine serum albumin, which does not diffuse through the plasma membrane, blocked ATP-induced [Ca(2+)](i), suggesting that estradiol acts at a membrane-associated estrogen receptor. Attenuation of [Ca(2+)](i) transients was mediated by estrogen action on VGCC. Nifedipine (10 microM), an L-type VGCC antagonist mimicked the effect of estrogen and when co-administered did not increase the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. N- and P-type VGCC antagonists omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (100 nM), attenuated the ATP-induced [Ca(2+)](i) transients. Co-administration of these blockers with estrogen induced a further decrease of the ATP-induced [Ca(2+)](i) flux. Together, these results suggest that although ATP stimulation of P2X receptors activates L-, N-, and P-type VGCC, estradiol primarily blocks L-type VGCC. The estradiol regulation of this ATP-induced [Ca(2+)](i) transients suggests a mechanism through which estradiol may modulate nociceptive signaling in the peripheral nervous system.

The Luteinizing Hormone Surge Is Preceded by an Estrogen-Induced Increase of Hypothalamic Progesterone in Ovariectomized and Adrenalectomized Rats

As circulating estrogen levels rise on the afternoon of proestrus, they stimulate the hypothalamo-pituitary axis. This estrogen positive feedback is pivotal to stimulate the luteinizing hormone (LH) surge required for ovulation and luteinization of ovarian follicles. In addition to estrogen, pre-LH surge progesterone is critical for an LH surge as was demonstrated by blocking progesterone synthesis. In ovariectomized (OVX) rats treated with trilostane, a blocker of the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) that catalyzes the conversion of pregnenolone to progesterone, estrogen did not induce an LH surge. Further, estrogen induced an LH surge in OVX and adrenalectomized (ADX) rats, indicating that the source of progesterone was neither the ovary nor adrenal gland. This estrogen-only LH surge was inhibited by pretreatment with trilostane, indicating that although the adrenal gland and ovary were not necessary for positive feedback, progesterone synthesis was critical for estrogen-induced positive feedback in an OVX/ADX rat. This suggested that the LH surge is dependent on the pre-LH surge synthesis of progesterone. Estrogen-induced progesterone receptors in the hypothalamus are vital for the LH surge, so a potential location for progesterone synthesis is the hypothalamus. OVX/ADX female rats were treated with 17β-estradiol (50 µg) and progesterone levels were assayed by RIA. Progesterone levels were elevated in hypothalamic tissue following estrogen treatment. No increases in tissue progesterone levels were found in parietal cortex, cerebellum, medulla, pituitary or plasma. Additionally, male rats that do not have an estrogen positive feedback-induced LH surge were examined. Castrated/ADX male rats had no increase in hypothalamic progesterone levels after estrogen treatment. Together, these data strongly suggest that estrogen enhances neuroprogesterone synthesis in the hypothalamus that is involved in the positive feedback regulating the LH surge.

Membrane Estrogen Receptors Stimulate Intracellular Calcium Release and Progesterone Synthesis in Hypothalamic Astrocytes

In hypothalamic astrocytes obtained from adult female rats, estradiol rapidly increased free cytoplasmic calcium concentrations ([Ca2+]i) that facilitate progesterone synthesis. The present study demonstrated that estradiol (1 nm) significantly and maximally stimulated progesterone synthesis within 5 min, supporting a rapid, nongenomic mechanism. The group I metabotropic glutamate receptor (mGluR1a) antagonist LY 367385 [(S)-(+)-a-amino-4-carboxy-2-methylbenzeneacetic acid] attenuated both the estradiol-induced [Ca2+]i release and progesterone synthesis. To investigate membrane-associated estrogen receptors (mERs), agonists for ERα, ERβ, STX-activated protein, and GPR30 were compared. The selective ERα agonist propylpyrazole triole (PPT) and STX most closely mimicked the estradiol-induced [Ca2+]i responses, where PPT was more potent but less efficacious than STX. Only high doses (100 nm) of selective ERβ agonist diarylpropionitrile (DPN) and GPR30 agonist G-1 induced estradiol-like [Ca2+]i responses. With the exception of DPN (even at 100 nm), all agonists stimulated progesterone synthesis. The PPT- and STX-induced [Ca2+]i release and progesterone synthesis were blocked by LY 367385. While the G-1-stimulated [Ca2+]i release was blocked by LY 367385, progesterone synthesis was not. Since GPR30 was detected intracellularly but not in the membrane, we interpreted these results to suggest that G-1 could activate mGluR1a on the membrane and GPR30 on the smooth endoplasmic reticulum to release intracellular calcium. Although STX and G-1 maximally stimulated [Ca2+]i release in astrocytes from estrogen receptor-α knock-out (ERKO) mice, estradiol in vivo did not stimulate progesterone synthesis in the ERKO mice. Together, these results indicate that mERα is mainly responsible for the rapid, membrane-initiated estradiol-signaling that leads to progesterone synthesis in hypothalamic astrocytes.

Estradiol-Induced Estrogen Receptor-α Trafficking

Estradiol has rapid actions in the CNS that are mediated by membrane estrogen receptors (ERs) and activate cell signaling pathways through interaction with metabotropic glutamate receptors (mGluRs). Membrane-initiated estradiol signaling increases the free cytoplasmic calcium concentration ([Ca2+]i) that stimulates the synthesis of neuroprogesterone in astrocytes. We used surface biotinylation to demonstrate that ERα has an extracellular portion. In addition to the full-length ERα [apparent molecular weight (MW), 66 kDa], surface biotinylation labeled an ERα-immunoreactive protein (MW, ∼52 kDa) identified by both COOH- and NH2-directed antibodies. Estradiol treatment regulated membrane levels of both proteins in parallel: within 5 min, estradiol significantly increased membrane levels of the 66 and 52 kDa ERα. Internalization, a measure of membrane receptor activation, was also increased by estradiol with a similar time course. Continuous treatment with estradiol for 24–48 h reduced ERα levels, suggesting receptor downregulation. Estradiol also increased mGluR1a trafficking and internalization, consistent with the proposed ERα–mGluR1a interaction. Blocking ER with ICI 182,780 or mGluR1a with LY 367385 prevented ERα trafficking to and from the membrane. Estradiol-induced [Ca2+]i flux was also significantly increased at the time of peak ERα activation/internalization. These results demonstrate that ERα is present in the membrane and has an extracellular portion. Furthermore, membrane levels and internalization of ERα are regulated by estradiol and mGluR1a ligands. The pattern of trafficking into and out of the membrane suggests that the changing concentration of estradiol during the estrous cycle regulates ERα to augment and then terminate membrane-initiated signaling.

Estrogen-Induced μ-Opioid Receptor Internalization in the Medial Preoptic Nucleus Is Mediated via Neuropeptide Y-Y1 Receptor Activation in the Arcuate Nucleus of Female Rats

The endogenous peptides β-endorphin (β-END) and neuropeptide Y (NPY) have been implicated in regulating sexual receptivity. Both β-END and NPY systems are activated by estrogen and inhibit female sexual receptivity. The initial estrogen-induced sexual nonreceptivity is correlated with the activation and internalization of μ-opioid receptors (MORs), in the medial preoptic nucleus (MPN). Progesterone reverses the estrogen-induced activation/internalization of MOR and induces the sexual receptive behavior lordosis. To determine whether NPY and endogenous opioids interact, we tested the hypothesis that estrogen-induced MOR activation is mediated through NPY-Y1 receptor (Y1R) activation. Retrograde tract tracing demonstrated Y1Ron β-END neurons that projected to the MPN. Sex steroid modulation of MOR in the MPN acts through NPY and the Y1R. Estradiol administration or intracerebroventricular injection of NPY activated/internalized Y1R in the arcuate nucleus and MOR in the MPN of ovariectomized (OVX) rats. Moreover, the selective Y1R agonist [Leu31, Pro34]-Neuropeptide Y (LPNY) internalized MOR in the MPN of OVX rats. The Y1R antagonist (Cys31, Nva34)-Neuropeptide Y (27–36)2 prevented estrogen-induced Y1R and MOR activation/internalization. NPY reversed the progesterone blockade of estradiol-induced Y1R and MOR internalization in the arcuate nucleus and MPN, respectively. Behaviorally, LPNY inhibited estrogen plus progesterone-induced lordosis, and the MOR-selective antagonist D-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr amide reversed LPNY-induced inhibition of lordosis. These results suggest that a sequential sex steroid activation of NPY and MOR circuits regulates sexual receptivity.