Fear potentiation and fear inhibition in a human fear-potentiated startle paradigm.

BACKGROUND: The inability to suppress excessive fear or anxiety is a significant clinical problem. In the laboratory, extinction is a preferred method for the study of fear inhibition; however, in this paradigm the same stimulus causes both elicitation (excitation) and inhibition of fear, making it difficult to know whether an experimental manipulation that affects extinction does so by affecting one or both of these processes. For this reason, we sought to develop a behavioral procedure in humans that would render a stimulus primarily inhibitory. METHODS: We adapted a conditional discrimination procedure (AX+/BX-), previously validated in animals, to a human fear-potentiated startle paradigm. Forty-one healthy volunteers were presented with one set of colored lights paired with the delivery of aversive airblasts to the throat (AX+) and a different series of lights presented without airblasts (BX-). RESULTS: Participants exhibited fear potentiation to AX+, discrimination between AX+ and BX-, and transfer of fear inhibition to A in an AB compound test but not in an AC compound test. CONCLUSIONS: We believe this procedure will advance clinical research on fear disorders, such as posttraumatic stress disorder and phobias, by providing an effective and relatively independent measure of fear potentiation and fear inhibition.     

ERalpha, but not ERbeta, mediates the expression of sexual behavior in the female rat

Estrogen has well known effects on sexual behavior, however the role of the estrogen receptors (ER) alpha and beta on sexual behavior remains to be fully determined. This study investigated the individual and co-operative involvement of ERalpha and beta on sexual behaviors in the adult female rat. Subtype selective ER agonists, propyl-pyrazole triol (PPT; ERalpha agonist) and diarylpropionitrile (DPN; ERbeta agonist) were utilized to examine each receptor subtype's contribution, individual and co-operative, for both receptive (lordosis) and proceptive (hopping/darting, 'ear wiggling') female sexual behaviors. Ovariectomized female rats received subcutaneous injections of either: sesame oil (OIL), dimethylsulfoxide (DMSO), estradiol benzoate (EB; 10 microg/0.1 ml OIL), one of three doses of the ERalpha agonist PPT (1.25mg, 2.5mg or 5.0mg/0.1 ml DMSO), one of three doses of the ERbeta agonist DPN (1.25mg, 2.5mg or 5.0mg/0.1 ml DMSO) or a combination dose of PPT and DPN (2.5mg PPT+2.5mg DPN/0.1 ml DMSO) for two consecutive days, 48 and 24h prior to testing followed by a progesterone injection (500 microg/0.1 ml OIL) 4h prior to testing in order to elicit sexual behavior. The ERalpha agonist PPT, but not the ERbeta agonist DPN, elicited both proceptive and receptive behavior. PPT at doses of 2.5 and 5.0mg significantly elicited lordosis and proceptive behavior ('ear wiggling', hopping and darting). Intriguingly, the administration of both agonists together at the 2.5mg dose resulted in reduced levels of proceptivity and receptivity, suggesting that ERbeta modulates ERalpha's ability to elicit receptive and proceptive sexual behavior.     

New roles for estrogen receptor beta in behavior and neuroendocrinology

In this review we critically examine the data on functions of the estrogen receptor beta (ERbeta) in both behavior and neuroendocrinology. The influence of estradiol via the ERbeta has been assessed using several methods: estrogen receptor knockout mice, specific ERbeta selective agonists, and phytoestrogens which preferentially bind to ERbeta rather than ERalpha. The behavior for which a solid database and consensus is forming is anxiety; activation of ERbeta reduces anxiety on a number of tasks and in several species. Moreover, the relationship between ERbeta and serotonin may be critical for the regulation of this behavior by estradiol. There have been very few studies on learning and memory but the little we know suggests that ERbeta is involved in visuospatial learning; in its absence learning is inhibited. Recent work has suggested a unique function for ERbeta in sexual differentiation; its activation in male neonates may promote defeminization of sexual behavior. Several neurotransmitter-containing neurons in the rat paraventricular nucleus coexpress ERbeta including; vasopressin, oxytocin, prolactin, and to a lesser extent corticotrophin releasing hormone. Given the potential for ERbeta to interact with these important neurotransmitters and its co-expression in gonadotropin releasing hormone neurons it is surprising how normal the hypothalamic-pituitary-adrenal and -gonadal axes appear to be in ERbeta knockout mice. Either this represents a species difference (the neuroanatomy has been conducted in the rat) or compensatory actions of ERalpha or other mechanisms. Exciting avenues for future research include; in vivo interactions between ERalpha and ERbeta, actions of non-estrogenic ligands with ERbeta, and the role of ERbeta in sexual differentiation.     

Estrogen deprivation and replacement modulate cerebral capillary density with vascular expression of angiogenic molecules in middle-aged female rats.

The effect of postmenopausal estrogen replacement therapy (ERT) on the risk or severity of cerebrovascular disorders is as yet unclear, and the evidence for flow preservation being a mechanism of estrogen neuroprotection remains elusive. The authors examined whether estrogen-mediated flow-preserving neuroprotective mechanisms, if any, may involve its angiogenic action. This study was conducted using middle-aged (44 weeks) female rats because of the importance of aging in cerebrovascular disease in women. Middle-aged female rats were subjected to sham operation, ovariectomy, or ovariectomy with ERT. The anatomic cerebral capillary morphology showed a significant reduction in the total capillary density in the frontal cortex after ovariectomy. This was associated with marked decreases in protein and gene expression of vascular endothelial growth factor and its angiogenic receptors in cerebral vessels, as demonstrated by immunohistochemistry and hybridization. The expression levels of both estrogen receptor (ER) subtypes, ERalpha and ERbeta, in cerebral vessels were significantly reduced after ovariectomy, but ERbeta was more dramatically downregulated as assessed by the ERbeta/ERalpha ratio. These ovariectomy-induced changes were completely prevented by ERT. Vascular endothelial growth factor appears to be a critical regulatory molecule for physiologic cerebral angiogenesis in middle-aged female rats and may play an important role in the flow-preserving neuroprotective action of estrogen through its angiogenic and antiapoptotic properties.     

Estrogen receptors alpha and beta mediate different aspects of the facilitatory effects of female cues on male risk taking

Male risk taking and decision making are affected by sex-related cues, with men making poorer and riskier decisions in the presence of females and, or their cues. In non-human species, female cues can also increase male risk taking, reducing their responses to predator threat. As estrogen receptors alpha and beta (ERalpha and ERbeta) are involved in the mediation of social and sexual responses, we investigated their roles in determining the effects of female-associated cues on male risk taking. We examined the effects of brief pre-exposure to the odors of either a novel or familiar estrous female on the avoidance of, and aversive responses to, predator threat (cat odor) in ERalpha and ERbeta wild type (alphaERWT, betaERWT) and gene-deleted (knockout, alphaERKO, betaERKO) male mice. Exposure of alphaERWT and betaERWT males to the odors of a novel, but not a familiar, estrous female mouse resulted in enhanced risk taking with the males displaying reduced avoidance of, and analgesic responses to, cat odor. In contrast, alphaERKO male mice failed to show any changes in risk taking, while betaERKO males, although displaying greater risk taking, did not distinguish between novel and familiar females, displaying similarly reduced avoidance responses to cat odor after exposure to either a novel or familiar female odor. These findings indicate that the gene for ERalpha is associated with the sexual mechanisms (response to estrous female) and the genes for ERbeta and ERalpha with the social (recognition of novel female) mechanisms underlying the effects of female cues on male risk taking.     

Estrogen receptor subtypes alpha and beta contribute to neuroprotection and increased Bcl-2 expression in primary hippocampal neurons

Estrogen receptor (ER) mediated neuroprotection has been demonstrated in both in vitro and in vivo model systems. However, the relative contribution by either ER subtype, ERalpha or ERbeta, to estrogen-induced neuroprotection remains unresolved. To address this question, we investigated the impact of selective ER agonists for either ERalpha, PPT, or ERbeta, DPN, to prevent neurodegeneration in cultured hippocampal neurons exposed to excitotoxic glutamate. Using three indicators of neuronal viability and survival, we demonstrated that both the ERalpha selective agonist PPT and the ERbeta selective agonist DPN protected hippocampal neurons against glutamate-induced cell death in a dose-dependent manner, with the maximal response occurring at 100 pM. Further analyses showed that both PPT and DPN enhanced Bcl-2 expression in hippocampal neurons, with an efficacy comparable to their neuroprotective capacity. Collectively, the present data indicate that activation of either ERalpha or ERbeta can promote neuroprotection in hippocampal neurons, suggesting that both receptor subtypes could be involved in estrogen neuroprotection. As ERbeta is highly expressed in the brain and has little or no expression in the breast or uterus, discovery and design of ERbeta selective molecules could provide a strategy for activating the beneficial effects of estrogen in the brain without activating untoward effects of estrogen in reproductive organs.     

Sex with knockout models: behavioral studies of estrogen receptor alpha.

Estrogens are an important class of steroid hormones, having multiple targets, in the body and brain, and exerting ubiquitous effects on behavior. At present, two estrogen receptors (ERalpha and beta) have been cloned and sequenced in mammals. In the brain these receptors are regionally specific, but both have widespread distributions, which are largely non-overlapping. Given the newly emerging complexities of estrogen's mechanisms of action it is important to distinguish which pathways are involved in modifying which behaviors. We use a knockout mouse, lacking functional copies of the estrogen receptor alpha (ERalpha) gene, to study the mechanisms by which estrogens mediate behaviors. There are pronounced ramifications of ERalpha gene disruption on behavior. First, female ERalpha knockout (ERalphaKO) mice do not display normal feminine sexual behavior. Second, treatment of adult mice with androgens promotes masculine sexual behavior in both sexes. However, male-typical sexual behavior is severely compromised in male and female ERalphaKOs. Third, male ERalphaKOs do not exhibit the same social preferences for female mice as do wildtype (WT) littermates. Thus, the ERalpha is essential for normal expression of sexual behaviors. In addition, gonadectomized ERalphaKO and WT mice rapidly learn to escape from the Morris water maze. Exogenous estrogen treatment prevents WT females from learning this task, yet, has no effect in ERalphaKO mice, suggesting that estrogens effects on learning in adult females involves the ERalpha. Based on these data we hypothesize that ERalpha mediates many of the effects of estrogen on sexual behavior, learning, and memory.     

Progestin receptor expression in the developing rat brain depends upon activation of estrogen receptor alpha and not estrogen receptor beta

Perinatal 17beta-estradiol (E2) rapidly and markedly affects the morphological and neurochemical organization of the vertebrate brain. For instance, the sex difference in perinatal progestin receptor (PR) immunoreactivity in the medial preoptic nucleus (MPN) of the rat brain is due to the intracellular conversion of testosterone into E2 in males. Neonatal alpha-fetoprotein prevents circulating estrogens from accessing the brain, therefore, to overcome alpha-fetoprotein sequestration of E2, estrogen replacement studies during development have used natural and synthetic estrogen dosages in the milligram to microgram range. These levels could be considered as supraphysiological. Moreover, it is not clear through which ER subtype E2 acts to induce PR expression in the neonatal rat MPN because E2 binds similarly to estrogen receptor (ER)alpha and ERbeta. Consequently, we investigated whether nanogram levels of E2 affected PR protein and mRNA levels in the neonatal MPN. Furthermore, propylpyrazole-triol (PPT), a highly selective agonist for ERalpha, and diarylpropionitrile (DPN), a highly selective agonist for ERbeta, were used to determine if E2-dependent PR expression in the neonatal rat is mediated through ERalpha and/or ERbeta. Immunocytochemistry and quantitative real-time RT-PCR determined that as little as 100 ng E2 significantly induced PR protein and mRNA in the female and neonatally castrated male MPN on PN 4, indicating that the neonatal rat brain is highly sensitive to circulating estrogens. PPT, but not DPN, induced PR expression in the neonatal MPN and arcuate nucleus (Arc), demonstrating that PR expression in the neonatal rat brain depends solely on E2 activated ERalpha. In the lateral bed nucleus of the stria terminalis (BSTL), neither PPT nor DPN affected PR expression, suggesting the presence of a gonadal hormone-independent PR regulatory mechanism.     

Estrogen receptor-alpha mediates estradiol attenuation of ATP-induced Ca2+ signaling in mouse dorsal root ganglion neurons

A mechanism underlying gender-related differences in pain perception may be estrogen modulation of nociceptive signaling in the peripheral nervous system. In rat, dorsal root ganglion (DRG) neurons express estrogen receptors (ERs) and estrogen rapidly attenuates ATP-induced Ca2+ signaling. To determine which estrogen receptor mediates rapid actions of estrogen, we showed ERalpha and ERbeta expression in DRG neurons from wild-type (WT) female mice by RT-PCR. To study whether ERalpha or ERbeta mediates this response, we compared estradiol action mediating Ca2+ signaling in DRG neurons from WT, ERalpha knockout (ERalphaKO), and ERbetaKO mice in vitro. ATP, an algesic agent, induced [Ca2+]i transients in 48% of small DRG neurons from WT mice. 17beta-Estradiol (E2) inhibited ATP-induced intracellular Ca2+ concentration ([Ca2+]i) with an IC50 of 27 nM. The effect of E2 was rapid (5-min exposure) and stereo specific; 17alpha-estradiol had no effect. E2 action was blocked by the ER antagonist ICI 182,780 (1 microM) in WT mouse. Estradiol coupled to bovine serum albumin (E-6-BSA), which does not penetrate the plasma membrane, had the same effect as E2 did, suggesting that a membrane-associated ER mediated the response. In DRG neurons from ERbetaKO mice, E2 attenuated the ATP-induced [Ca2+]i flux as it did in WT mice, but in DRG neurons from ERalphaKO mice, E2 failed to inhibit the ATP-induced [Ca2+]i increase. These results show that mouse DRG neurons express ERs and the rapid attenuation of ATP-induced [Ca2+]i signaling is mediated by membrane-associated ERalpha.     

Regionally specific modulation of brain apolipoprotein E in the mouse during the estrous cycle and by exogenous 17beta estradiol

Studies have suggested that 17beta estradiol (E2) can modify apolipoprotein E (apoE) expression. The current study determined if apoE protein varied in different regions of the mouse brain as a function of the estrous cycle and if E2 could increase apoE protein expression. In this study apoE concentration was lowest on estrus in the hippocampus, cingulate cortex and frontal cortex. In contrast, apoE concentration was highest on estrus in the olfactory bulb and cerebellum. There were no differences in the striatal apoE expression throughout the estrous cycle. Exogenous E2 significantly raised tissue levels of apoE in the olfactory bulb and cerebellum at 5 days after treatment. There was a slight, but nonsignificant increase in cortical expression of apoE and no change in striatum. Immunocytochemical localization studies found estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in cortical neurons and glia. In the cerebellum and olfactory bulb, ERbeta was seen primarily in glia. ERalpha was not observed in the cerebellum and was rare in the olfactory bulb. Neither ERalpha nor ERbeta was seen in the striatum. Our data show regional differences in the production of apoE throughout the estrous cycle. In addition, exogenous E2 has regionally specific effects on apoE expression. Regional variability in apoE production appears to vary as a function of the estrogen receptor subtype.     

Distribution of estrogen receptor alpha and beta in the mouse central nervous system: in vivo autoradiographic and immunocytochemical analyses

Although the distribution of estrogen receptor beta (ERbeta) immunoreactivity in the rat central nervous has been reported, no such data are available in the mouse. The present study used in vivo autoradiography utilizing a (125)I-estrogen that has equal binding affinity for both receptors as well as immunohistochemistry for ERbeta and ERalpha, to investigate and compare the distribution of the two ERs in the mouse CNS. The use specific antisera against ERalpha and ERbeta allowed us to evaluate the contribution of these receptors to the binding detected with autoradiography. In addition, data were collected in ovariectomized wildtype and ERalpha KO (knockout) mice to examine developmental regulation of ERbeta expression by ERalpha. These studies revealed that in the mouse CNS, combining immunoreactivity for ERalpha with that for ERbeta accounted for all regions where binding was seen using autoradiography. Therefore, these data strongly suggest that the major contributors of estrogen binding in the mouse CNS are ERalpha and ERbeta. Together, these data provide an anatomical foundation for future studies and advance our understanding of estrogen action in the CNS. Moreover, since the immunocytochemical images were similar in wildtype and ERalpha KO mice, these studies suggest that the lack of ERalpha does not influence the expression of ERbeta in the central nervous system.     

6-Hydroxydopamine lesion in thalamic reticular nucleus reduces anxiety behaviour in the rat

Serotonin (5-HT) is known to play a role in the suppression of the lordosis response in males. We have previously shown that there is a sex difference in the density of 5-HT immunoreactive (5-HT-ir) fibers in the ventrolateral division of the adult ventromedial nucleus of the hypothalamus (VMNvl) and that neonatal administration of estradiol (E2) increases 5-HT-ir in the female VMNvl to male-typical levels. Here we demonstrate that postnatal administration of the ERalpha agonist 1,3,5-tris(4-Hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), but not the ERbeta agonist diarylpropionitrile (DPN), also masculinizes 5-HT-ir in the female VMNvl, suggesting a mechanistic role for ERalpha in this process. Sexual receptivity, as ascertained by the lordosis quotient, was unaffected by either PPT or DPN treatment but nearly abolished by estradiol benzoate (EB), a synthetic estrogen with high affinity for both ERalpha and ERbeta. Collectively, these observations show that postnatal estrogens increase the density of 5-HT projections to the VMNvl via an ERalpha dependent mechanism, but that this increased inhibitory input is not sufficient to suppress the lordosis response.     

Expression of estrogen receptor -alpha and -beta immunoreactivity in the cultured neonatal suprachiasmatic nucleus: with special attention to GABAergic neurons.

This study investigated the expression patterns of estrogen receptor -alpha (ERalpha) and -beta (ERbeta) in cultured cells of the suprachiasmatic nucleus (SCN) in neonatal rats by combined application of cell culture and double-label immunocytochemistry. The results revealed that the immunoreactivity for either ERalpha or ERbeta (with predominance of ERbeta) was localized in not only neurons but also astrocytes. The co-expression of both ERalpha and ERbeta in the same individual cell was also demonstrated by the double-label immunocytochemistry. The observations also provide a direct evidence for the differential expression of ER subtypes within GABAergic SCN neurons in vitro and suggest that estrogen's effect on the SCN may be mediated at least in part by its ER-containing GABAergic neurons.     

Differential effects of ovarian steroids on anxiety versus fear as measured by open field test and fear-potentiated startle

The ovarian steroids, estrogen (E) and progesterone (P), have been shown to affect anxiety and fear in humans and animals, although with inconsistent results. These ambiguous findings may be due to differential actions of ovarian steroids on anxiety versus fear. To investigate such a role, we used the open field test (OFT) and fear-potentiated startle (FPS). We examined these behaviors between cycling female rats in proestrus (high E and rising P) or diestrus (low E and P), as well as between ovariectomized rats treated for 2 weeks with placebo, E, or E plus P (OVX, OVX/E, OVX/EP, respectively). We found no differences in anxiety-like or fear behaviors in OFT or FPS between proestrus and diestrus rats, perhaps due to the opposing effects of E and P. In contrast, we found that the OVX/E rats spent more time in the center of the OFT compared to the OVX and OVX/EP rats with no difference in overall activity level, suggesting that E reduced anxiety and this was opposed by P. With FPS, the OVX/E rats showed increased startle in the first third of the testing session, followed by a rapid decline in startle magnitude in subsequent trials. The addition of P to E treatment counteracted this effect. In conclusion, E may have differential effects on specific components of anxiety and fear; E may decrease anxiety in a naturalistic environment, but intensify both fear learning and extinction processes. P antagonizes these E effects on anxiety and fear.     

Changes in estrogen receptor-alpha mRNA in the mouse cortex during development

Estrogen plays a critical role in brain development and is responsible for generating sex differences in cognition and emotion. Studies in rodent models have shown high levels of estrogen binding in non-reproductive areas of the brain during development, including the cortex and hippocampus, yet binding is diminished in the same areas of the adult brain. These binding studies demonstrated that estrogen receptors decline in the cortex during development but did not identify which of the two estrogen receptors was present. In the current study, we examined the expression of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in the mouse cortex during the first month of life. Messenger RNA was isolated from cortical tissue taken from C57BL/6 mice on postnatal day (PND) 1, 4, 10, 18 and 25 and expression levels were determined by real-time PCR. ERalpha mRNA expression in the mouse cortex at PND 25 was significantly reduced as compared to PND 1 (p<0.01). ERbeta mRNA expression at PND 25 was significantly increased as compared to PND 1 (p<0.05). Although the increase in ERbeta mRNA was statistically significant, the ERbeta levels were extremely low in the isocortex compared to ERalpha mRNA levels, suggesting that ERalpha may play a more critical role in the developmental decrease of estradiol binding than ERbeta. Additionally, we measured ERalpha mRNA expression in organotypic explant cultures of cortex taken from PND 3 mice. Explants were maintained in vitro for 3 weeks. mRNA was isolated at several time points and ERalpha and ERbeta mRNA was measured by real-time RT-PCR. ERalpha and ERbeta mRNA levels reflected a similar pattern in vitro and in vivo, suggesting that signals outside the cortex are not needed for this developmental change. This study lays the groundwork for an understanding of the mechanisms of the developmental regulation of ERalpha mRNA.     

Chronic estrogenic drug treatment increases preproenkephalin mRNA levels in the rat striatum and nucleus accumbens

Estrogens modulate the expression of preproenkephalin (PPE) in the hypothalamus but little is known for other brain regions. The present study investigated the effect of hormonal withdrawal and replacement therapy on PPE expression in the striatum, nucleus accumbens and cortex. Ovariectomized Sprague-Dawley rats were treated for 2 weeks with estradiol, a specific ligand for estrogen receptor alpha (ERalpha), 4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) and estrogen receptor beta (ERbeta) 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), or the selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene. Brain PPE mRNA levels, measured by in situ hybridization, were high in the striatum and nucleus accumbens compared to the low expression in the cortex. Ovariectomy decreased uterine weights compared to intact uterus, which was corrected by estradiol and PPT. Tamoxifen and raloxifene partially stimulated uterine weights while DPN left it unchanged. In the anterior, median and posterior striatum and in the core and shell of the nucleus accumbens, ovariectomy decreased PPE mRNA levels compared to intact rats, this was corrected by estradiol treatment except for the posterior striatum. PPT, DPN, tamoxifen and raloxifene reproduced the estradiol effect. In the prefrontal and cingulate cortices, neither ovariectomy nor treatments changed PPE mRNA levels. These results show for the first time that estradiol increases PPE mRNA in the striatum and nucleus accumbens. This effect is observed also with estrogen receptor agonists for the ERalpha and ERbeta as well as with SERMs.