Ovarian hormone loss impairs excitatory synaptic transmission at hippocampal CA3-CA1 synapses.

Premature and long-term ovarian hormone loss following ovariectomy (OVX) is associated with cognitive impairment. This condition is prevented by estradiol (E2) therapy when initiated shortly following OVX but not after substantial delay. To determine whether these clinical findings are correlated with changes in synaptic functions, we used adult OVX rats to evaluate the consequences of short-term (7-10 d, OVXControl) and long-term (∼5 months, OVXLT) ovarian hormone loss, as well as subsequent in vivo E2 treatment, on excitatory synaptic transmission at the hippocampal CA3-CA1 synapses important for learning and memory. The results show that ovarian hormone loss was associated with a marked decrease in synaptic strength. E2 treatment increased synaptic strength in OVXControl but not OVXLT rats, demonstrating a change in the efficacy for E2 5 months following OVX. E2 also had a more rapid effect: within minutes of bath application, E2 acutely increased synaptic strength in all groups except OVXLT rats that did not receive in vivo E2 treatment. E2's acute effect was mediated postsynaptically, and required Ca(2+) influx through the voltage-gated Ca(2+) channels. Despite E2's acute effect, synaptic strength of OVXLT rats remained significantly lower than that of OVXControl rats. Thus, changes in CA3-CA1 synaptic transmission associated with ovarian hormone loss cannot be fully reversed with delayed E2 treatment. Given that synaptic strength at CA3-CA1 synapses is related to the ability to learn hippocampus-dependent tasks, these findings provide additional insights for understanding cognitive impairment-associated long-term ovarian hormone loss and ineffectiveness for delayed E2 treatment to maintain cognitive functions.

Prenatal stress generates deficits in rat social behavior: Reversal by oxytocin.

Neurodevelopmental changes induced by environmental stress exposure play a significant but poorly defined role in the etiology of schizophrenia. Exposure of pregnant female rats to a series of unpredictable stresses during the final week of pregnancy generates behavioral deficits and molecular changes in the offspring similar to those observed in schizophrenic individuals. We used this rat prenatal stress preparation to investigate social withdrawal behaviors that may have relevance to the negative symptoms of schizophrenia. The cumulative time adult male offspring of stress-exposed pregnant female rats actively interacted with a weight-matched, same-sex peer was decreased approximately 76% relative to non-stress exposed control rats. Prenatal stress exposure also diminished the quality of the social interaction behavior indicative of reduced social drive. Analysis of the oxytocinergic system in the prenatally stressed male rats revealed significantly less oxytocin mRNA in the paraventricular nucleus and increased oxytocin receptor binding in the central amygdala. Moreover, oxytocin, but not vasopressin, administration into the central amygdala reversed the social incompetence of the prenatally stressed rats without increasing behavior in non-stressed control animals. In addition, cross-fostering pups from prenatally stressed mothers to non-stressed mothers failed to improve the social deficit of the prenatally stressed male offspring. Two behavioral assays designed to measure anxiety did not differentiate the prenatally stressed rats from non-stressed controls. These data indicate that prenatal stress may be an etiologically appropriate animal model for some aspects of schizophrenic social withdrawal. Furthermore, unpredictable prenatal stress exposure selectively degrades social interaction behaviors without increasing anxiety measures.

Estradiol reduces nonclassical transcription at cyclic adenosine 3',5'-monophosphate response elements in glioma cells expressing estrogen receptor alpha.

Estradiol can protect the brain from a variety of insults by activating membrane-initiated signaling pathways, and thereby modulate gene expression and lead to functional changes in neurons. These direct neuronal effects of the hormone have been well documented; however, it is less understood what effects estradiol may have on nonneuronal cells of the central nervous system. There is evidence that estradiol levels can induce the release of glial-derived growth factors and other cytokines, suggesting that estradiol may both directly and indirectly protect neurons. To determine whether 17beta-estradiol (E2) can activate rapid signaling and modulate nonclassical transcription in astrocytes, we stably transfected the C6 rat glioblastoma cell line with human estrogen receptor (ER) alpha (C6ERalpha) or rat ERbeta (C6ERbeta). Introduction of a cAMP response element-luciferase reporter gene into C6, C6ERalpha, and C6ERbeta cells leads to the observation that E2 treatment reduced isoproterenol-stimulated luciferase activity by 35% in C6ERalpha but had no effect on reporter gene expression in C6ERbeta or untransfected C6 cells. A similar effect was seen with a membrane-impermeable estrogen (E2-BSA), suggesting the modulation of nonclassical transcription by estradiol treatment is mediated by the activation of a membrane-initiated signaling pathway. Furthermore, pretreatment with wortmannin (phosphatidylinsositol 3-kinase) or U73122 (phospholipase C) attenuated the E2-induced reduction in nonclassical transcription. We conclude that E2 treatment reduces cAMP response element-mediated transcription in glioma cells expressing ERalpha and that this reduction is dependent on the activation of membrane-initiated signaling. These findings suggest a novel model of estrogen rapid signaling in astrocytes that leads to modulation of nonclassical transcription.

Social interaction deficits caused by chronic phencyclidine administration are reversed by oxytocin.

Chronic administration of phencyclidine (PCP) has been advanced as a valid animal model of the social deficit symptoms of schizophrenia. In these studies, the cumulative time that male rats treated once a day for 14 days with PCP actively engaged in social behavior was decreased approximately 75% relative to saline-treated control animals. In addition, these socially impaired rats had an increase in the relative amount of noncontact interactions compared with saline-injected peers. Social behaviors were preferentially affected by PCP treatment because in two anxiety-related behavioral assays, the open field and light/dark emergence tests, there was a failure to differentiate between the PCP-treated rats and saline-injected control rats. Considering the general importance of the neuropeptides oxytocin and vasopressin in male social behaviors, studies of molecular markers related to these neuropeptides were performed. Hypothalamic oxytocin mRNA expression was significantly decreased while oxytocin receptor binding was increased in the central nucleus of the amygdala following chronic PCP treatment. Given the significance of central nucleus of the amygdala in social behavior, oxytocin was infused into the central nucleus of experimental and control male rats, and their postinfusion social interaction and open field behaviors were analyzed. A bilateral infusion of 1 mug of oxytocin into the central amygdala selectively restored the normal quantity and quality of social behavior in chronic PCP-treated male rats without altering open field behaviors. These findings suggest that deficits in the central oxytocinergic system may underlie the social impairment exhibited in this animal model of schizophrenia.

Estrogen activation of cyclic adenosine 5'-monophosphate response element-mediated transcription requires the extracellularly regulated kinase/mitogen-activated protein kinase pathway.

The ability of estrogen to rapidly initiate a variety of signal transduction cascades is increasingly recognized as playing an important role in a number of tissue-specific transcriptional actions of the hormone. In vivo, estrogen rapidly elicits phosphorylation of cAMP response element-binding protein (CREB). We have previously shown that both ER alpha and ER beta are capable of activating the MAPK pathway in response to a low dose of 17beta-estradiol. In the present study, the ability of estrogen to act through both ER alpha and ER beta to increase CREB phosphorylation was evaluated in an immortalized hippocampal cell line stably expressing either receptor. Estrogen treatment promoted rapid CREB phosphorylation, reaching a maximum by 15 min. This activation is completely blocked by the antiestrogen ICI 182,780, suggesting an estrogen receptor-dependent mechanism. The addition of the mitogen/ERK kinase-1 inhibitor, PD98059, also blocked the ability of estrogen to signal to CREB phosphorylation. Estrogen also caused an increase in p90Rsk activity, a critical mediator of MAPK effects. Surprisingly, blockade of the protein kinase A pathway in cells treated with estrogen did not affect estrogen-mediated CREB phosphorylation. Thus, MAPK and p90Rsk appear to be the primary mediators of estrogen-induced gene transcription through ER alpha and ER beta.

Estrogen receptor-mediated neuroprotection from oxidative stress requires activation of the mitogen-activated protein kinase pathway.

It is well documented that estrogen mediates responses by both genomic and nongenomic mechanisms, both of which are important for cell survival. Because direct evidence showing that the estrogen receptors (ERs) alpha and/or beta can activate rapid signaling that may mediate neuroprotection is lacking, the hippocampal-derived cell line, HT22, was stably transfected with ERalpha (HTERalpha), ERbeta (HTERbeta), or a mutated form of ERalpha (HTERalphaHE27), which lacks the ability to mediate ER element-mediated transcription. Treatment of HT22, HTERalpha, HTERbeta, and HTERalphaHE27 cells with glutamate (5 mM) resulted in a significant decrease in cell viability. Pretreatment for 15 min with 10 nM 17beta-estradiol resulted in a 50% increase in the number of living cells in HTERalpha and HTERbeta cells but not in HT22 cells. The ER antagonist ICI 182,780 and the MEK inhibitor PD98059 prevented 17beta-estradiol-mediated protection. In HTERalphaHE27 cells, 17beta-estradiol rapidly phosphorylated ERK2 (within 15 min), in the absence of estrogen response element-mediated transcription. Treatment of HTERalphaHE27 cells with 10 nM 17beta-estradiol partially reversed the cell death produced by glutamate treatment. This study demonstrates that activation of either ERalpha or ERbeta can result in neuroprotection and that activation of the MAPK pathway is an important part of the neuroprotective mechanism.

Tamoxifen enhances choline acetyltransferase mRNA expression in rat basal forebrain cholinergic neurons.

Novel estrogen-like molecules known as SERMs (selective estrogen receptor modulators) produce many of the beneficial estrogen-like actions without the detrimental side-effects. The SERM, tamoxifen, an estrogen-like molecule with both agonist and antagonist properties, is widely prescribed for the treatment of breast cancer. While the effects of tamoxifen are being evaluated in many peripheral tissues, its effects in the central nervous system (CNS) have been largely ignored. In the present study, we begin to evaluate the effects of tamoxifen in the rat basal forebrain, a region known to be highly responsive to estrogen. We compared the effects of short-term (24 h) tamoxifen treatment to that of estrogen on ChAT mRNA expression in cholinergic neurons. In addition, we examined the effect of tamoxifen in the presence and absence of estrogen. Our results indicate that tamoxifen enhances ChAT expression in a manner similar to that of estrogen in several basal forebrain regions. In contrast, tamoxifen exhibits antagonist properties with respect to estrogen-induction of progesterone receptor mRNA in the medial preoptic nucleus. These results indicate tamoxifen has estrogenic properties with respect to cholinergic neurons, suggesting a previously unidentified effect of this agent in the CNS.

Endogenous neurotensin attenuates dopamine-dependent locomotion and stereotypy.

The neuropeptide neurotensin (NT) is highly sensitive to changes in dopaminergic signaling in the striatum, and is thought to modulate dopamine-mediated behaviors. To explore the interaction of NT with the dopamine system, we utilized mice with a targeted deletion of dopamine synthesis specifically in dopaminergic neurons. Dopamine levels in dopamine-deficient (DD) mice are less than 1% of control mice, and they require daily administration of the dopamine precursor L-dihydroxyphenylalanine (L-DOPA) for survival. DD mice are supersensitive to the effects of dopamine, becoming hyperactive relative to control mice in the presence of L-DOPA. We show that 24 h after L-DOPA treatment, when DD mice are in a "dopamine-depleted" state, Nt mRNA levels in the striatum of DD mice are similar to those in control mice. Administration of L-DOPA or L-DOPA plus the L-amino acid decarboxylase inhibitor, carbidopa, (C/L-DOPA) induced Nt expression in the striatum of DD mice. The dopamine D1 receptor antagonist, SCH23390, blocked C/L-DOPA-induced Nt. To test the hypothesis that this striatal Nt expression modulated dopamine-mediated behavior in DD mice, we administered SR 48692, an antagonist of the high affinity NT receptor, together with L-DOPA or C/L-DOPA. L-DOPA-induced hyperlocomotion and C/L-DOPA-induced stereotypy were potentiated by peripheral administration of SR 48692. Furthermore, intrastriatal microinjections of SR 48692 augmented L-DOPA-induced hyperlocomotion. These results demonstrate a dynamic regulation of striatal Nt expression by dopamine via D1 receptors in DD mice, and point to a physiological role for endogenous striatal NT in counteracting motor behaviors induced by an overactive dopamine system.

17beta-Estradiol and the phytoestrogen genistein attenuate neuronal apoptosis induced by the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin.

Estrogenic compounds have been shown to protect neurons from a variety of toxic stimuli in vitro and in vivo and depletion of estrogen at menopause has been associated with increased risk of neurodegenerative diseases. Genistein is an isoflavone soy derivative that binds to estrogen receptors with selective estrogen receptor modulator (SERM) properties. Recent FDA recommendations of soy intake for cholesterol reduction have prompted investigation into the potentially estrogenic role of dietary soy phytochemicals in the brain. In this study, we have shown that 50nM genistein significantly reduces neuronal apoptosis in an estrogen receptor-dependent manner. The importance of apoptosis in the brain has been recognized with regard to organization of the developing brain as well as degeneration in response to disease or stroke; however, the effects of estrogenic compounds on neuronal apoptosis have not been thoroughly examined. We developed a model of apoptotic toxicity in primary cortical neurons by using the endoplasmic reticulum (ER) calcium-ATPase inhibitor, thapsigargin, to test potential anti-apoptotic effects of 17beta-estradiol and genistein. Estrogen receptor beta, but not estrogen receptor alpha, was detected in our primary neuron cultures. Thapsigargin-induced apoptosis was confirmed by loss of mitochondrial function, DNA laddering, nuclear condensation and fragmentation, and caspase activation. Both 17beta-estradiol and genistein reduced the number of apoptotic neurons and reduced the number of neurons containing active caspase-3. This effect was blocked by co-addition of ICI 182780. Our results demonstrate that genistein and 17beta-estradiol have comparable anti-apoptotic properties in primary cortical neurons and that these properties are mediated through estrogen receptors.

Hypothalamic expression of Eap1 is not directly controlled by ovarian steroids.

A gene termed EAP1 (enhanced at puberty 1) was recently identified as a transcriptional regulator of female neuroendocrine reproductive function. We have now used in vivo and in vitro assays, and the female rat as an animal model, to determine whether Eap1 gene expression is regulated by ovarian steroids. Eap1 mRNA abundance decreases in both the hypothalamus and cerebral cortex during the infantile-juvenile phases of development, but it increases selectively in the hypothalamus at puberty, suggesting that in contrast to the general decline in expression observed in immature animals, the region-specific increase in Eap1 mRNA levels that occurs at puberty might be elicited by ovarian steroids. This is, however, not the case, because hypothalamic Eap1 mRNA levels increase at the expected time of puberty in rats ovariectomized at the beginning of the juvenile period. Although a subpopulation of EAP1-containing cells in the medial basal hypothalamus (MBH) and preoptic area express estrogen receptor-alpha (ERalpha), the 5'-flanking region of the rat Eap1 (rEap1) gene does not contain a complete estrogen-responsive element, and no such estrogen-responsive element is detected within 100 kb of the rEap1 locus. Functional promoter assays showed that neither estradiol (E(2)) alone nor a combination of E(2) plus progesterone increases rEap1 gene transcription. Likewise, E(2) administered to ovariectomized immature rats elicited a robust surge of LH but increased neither preoptic area nor MBH Eap1 mRNA levels. E(2)/progesterone-treated rats showed a massive elevation in plasma LH but only a modest increase in Eap1 mRNA levels, limited to the MBH. These results indicate that hypothalamic Eap1 expression is not directly controlled by ovarian steroids and suggest that Eap1 expression increases at puberty driven by ovary-independent, centrally initiated events.

Multiple pathways transmit neuroprotective effects of gonadal steroids.

Numerous preclinical studies suggest that gonadal steroids, particularly estrogen, may be neuroprotective against insult or disease progression. This paper reviews the mechanisms contributing to estrogen-mediated neuroprotection. Rapid signaling pathways, such as MAPK, PI3K, Akt, and PKC, are required for estrogen's ability to provide neuroprotection. These rapid signaling pathways converge on genomic pathways to modulate transcription of E2-responsive genes via ERE-dependent and ERE-independent mechanisms. It is clear that both rapid signaling and transcription are important for estrogen's neuroprotective effects. A mechanistic understanding of estrogen-mediated neuroprotection is crucial for the development of therapeutic interventions that enhance quality of life without deleterious side effects.

Estrogen-mediated neuroprotection against beta-amyloid toxicity requires expression of estrogen receptor alpha or beta and activation of the MAPK pathway.

It is well documented that estrogen can activate rapid signaling pathways in a variety of cell types. These non-classical effects of estrogen have been reported to be important for cell survival after exposure to a variety of neurotoxic insults. Since direct evidence of the ability of the estrogen receptors (ERs) alpha and/or beta to mediate such responses is lacking, the hippocampal-derived cell line HT22 was stably transfected with either ERalpha (HTERalpha) or ERbeta (HTERbeta). In HTERalpha and HTERbeta cells, but not untransfected cells, an increase in ERK2 phosphorylation was measured within 15 min of 17beta-estradiol treatment. The ER antagonist ICI 182, 780 (1 microm) and the MEK inhibitor, PD98059 (50 microm) blocked this increase in ERK2 phosphorylation. Treatment of HT22, HTERalpha and HTERbeta cells with the beta-amyloid peptide (25-35) (10 micro m) resulted in a significant decrease in cell viability. Pre-treatment for 15 min with 10 nm 17beta-estradiol resulted in a 50% increase in the number of living cells in HTERalpha and HTERbeta cells, but not in HT22 cells. Finally, ICI 182, 780 and PD98059 prevented 17beta-estradiol-mediated protection. This study demonstrates that both ERalpha and ERbeta can couple to rapid signaling events that mediate estrogen-elicited neuroprotection.