Carboxypeptidase E protects hippocampal neurons during stress in male mice by up-regulating prosurvival BCL2 protein expression.

Prolonged chronic stress causing elevated plasma glucocorticoids leads to neurodegeneration. Adaptation to stress (allostasis) through neuroprotective mechanisms can delay this process. Studies on hippocampal neurons have identified carboxypeptidase E (CPE) as a novel neuroprotective protein that acts extracellularly, independent of its enzymatic activity, although the mechanism of action is unclear. Here, we aim to determine if CPE plays a neuroprotective role in allostasis in mouse hippocampus during chronic restraint stress (CRS), and the molecular mechanisms involved. Quantitative RT-PCR/in situ hybridization and Western blots were used to assay for mRNA and protein. After mild CRS (1 h/d for 7 d), CPE protein and mRNA were significantly elevated in the hippocampal CA3 region, compared to naïve littermates. In addition, luciferase reporter assays identified a functional glucocorticoid regulatory element within the cpe promoter that mediated the up-regulation of CPE expression in primary hippocampal neurons following dexamethasone treatment, suggesting that circulating plasma glucocorticoids could evoke a similar effect on CPE in the hippocampus in vivo. Overexpression of CPE in hippocampal neurons, or CRS in mice, resulted in elevated prosurvival BCL2 protein/mRNA and p-AKT levels in the hippocampus; however, CPE(-/-) mice showed a decrease. Thus, during mild CRS, CPE expression is up-regulated, possibly contributed by glucocorticoids, to mediate neuroprotection of the hippocampus by enhancing BCL2 expression through AKT signaling, and thereby maintaining allostasis.

Gonadotropin-releasing hormone neurons express estrogen receptor-beta.

CONTEXT: Recent identification of the second estrogen receptor (ER) isoform (ER-beta) within GnRH neurons of the rodent brain has generated much enthusiasm in the field of neuroendocrine research by questioning the dogma that GnRH cells do not directly sense changes in circulating estrogens. OBJECTIVE: To address the issue of whether GnRH neurons of the human hypothalamus also contain ER-beta, we have performed dual-label immunocytochemical studies. DESIGN: Tissue sections were prepared from autopsy samples of male human individuals (n = 8; age < 50 yr), with sudden causes of death. Technical efforts were made to minimize postmortem interval (<24 h), optimize tissue fixation (use of a mixture of 2% paraformaldehyde and 4% acrolein for four tissue samples), and sensitize the immunocytochemical detection of ER-beta (application of silver-intensified nickel-diaminobenzidine chromogen). MAIN OUTCOME MEASURE: Distribution and percent ratio of GnRH neurons that also contained ER-beta immunoreactivity were analyzed under the light microscope. RESULTS: With acrolein in tissue fixative, nuclear ER-beta immunoreactivity was observed in 10.8-28.0% of GnRH neurons of the four different individuals. ER-beta-containing GnRH neurons were widely distributed in the hypothalamus, without showing a noticeable preference in regional location. CONCLUSIONS: The demonstration of ER-beta and the previous lack of detection of ER-alpha in human GnRH cells indicate that estrogens may exert direct actions upon GnRH neurons exclusively through ER-beta. In the light of differing ligand-binding characteristics of ER-beta from those of ER-alpha, this discovery offers a potential new approach to influence estrogen feedback to GnRH neurons through ER-beta-selective receptor ligands.

Three-dimensional representation of the neurotransmitter systems of the human hypothalamus: inputs of the gonadotrophin hormone-releasing hormone neuronal system.

The gonadotrophin-releasing hormone (GnRH) represents the final common pathway of a neuronal network that integrates multiple external and internal factors to control fertility. Among the many inputs GnRH neurones receive, oestrogens play the most important role. In females, oestrogen, in addition to the negative feedback, also exhibits a positive feedback influence upon the activity and output of GnRH neurones to generate the preovulatory luteinising hormone surge and ovulation. Until recently, the belief has been that the GnRH neurones do not contain oestrogen receptors and that the action of oestrogen upon GnRH neurones is indirect, involving several, oestrogen-sensitive neurotransmitter and neuromodulator systems that trans-synaptically regulate the activity of the GnRH neurones. Although this concept still holds for humans, recent studies indicate that oestrogen receptor-beta is expressed in GnRH neurones of the rat. This review provides three dimensional stereoscopic images of GnRH-immunoreactive (IR) and some peptidergic (neuropeptide Y-, substance P-, beta-endorphin-, leu-enkaphalin-, corticotrophin hormone-releasing- and galanin-IR) and catecholaminergic neurones and the communication of these potential oestrogen-sensitive neuronal systems with GnRH neurones in the human hypothalamus. Because the post-mortem human tissue does not allow the electron microscopic identification of synapses on GnRH neurones, the data presented here are based on light microscopic immunocytochemical experiments using high magnification with oil immersion, semithin sections or confocal microscopy.

Estrogen stimulates galanin expression within luteinizing hormone-releasing hormone-immunoreactive (LHRH-i) neurons via estrogen receptor-beta (ERbeta) in the female rat brain.

Among the many factors that integrate the activity of the luteinizing hormone-releasing hormone (LHRH) neuronal system, estrogens play the most important role. Until recently, the belief has been that the LHRH neurons do not contain estrogen receptors and that the action of estrogen upon LHRH neurons is indirect involving several, estrogen-sensitive neurotransmitter and neuromodulator systems that regulate the activity of the LHRH neurons. Based on our recent findings that LHRH neurons of the female rat co-express galanin, that galanin is a potent LHRH releasing peptide, and that estrogen receptor-beta (ERbeta) is present in LHRH neurons, we have evaluated the effect of 17beta-estradiol on the expression of galanin within LHRH neurons. By combining immunocytochemistry for LHRH and in situ hybridization histochemistry for galanin, we demonstrate that 17beta-estradiol stimulates galanin expression within 2h following their administration to ovariectomized rats. Maximal expression, however, required a longer treatment regimen (3 days). These observations strongly suggest that estrogens stimulate galanin expression within LHRH neurons directly, via ERbeta. Moreover, ERbeta may mediate, at least in part, the positive feedback effect of estrogens during the preovulatory LHRH and subsequent LH surges.

Bi-directional associations between galanin and luteinizing hormone-releasing hormone neuronal systems in the human diencephalon.

Evidence suggests that galanin plays an important role in the regulation of reproduction in the rat. Galanin is colocalized with luteinizing hormone (LH)-releasing hormone (LHRH) in a subset of LHRH neurons in female rats and galanin-immunoreactive (galanin-IR) nerve terminals innervate LHRH neurons. Recent studies indicate that galanin may control gonadal functions in rats at two different levels: (i) via direct modulation of pituitary LH secretion and/or (ii) indirectly via the regulation of the hypothalamic LHRH release. However, the morphological substrate of any similar modulation is not known in human. In the present series of experiments we first mapped the galanin-IR and LHRH-IR neural elements in human brain, utilizing single label immunohistochemistry. Then, following the superimposition of the maps of these systems, the overlapping sites were identified with double labeling immunocytochemistry and examined in order to verify the putative juxtapositions between galanin-IR and LHRH-IR structures. LHRH and galanin immunoreactivity were detected mainly in the medial basal hypothalamus, in the medial preoptic area and along the diagonal band of Broca. Careful examination of the IR elements in the overlapping areas revealed close, bi-directional contacts between galanin-IR and LHRH-IR structures, which have been verified in semithin plastic sections. These galanin-LHRH and LHRH-galanin juxtapositions were most numerous in the medial preoptic area and in the infundibulum/median eminence of the human diencephalon. In conclusion, the present study is the first to reveal bi-directional juxtapositions between galanin- and LHRH-IR neural elements in the human diencephalon. These galanin-LHRH and LHRH-galanin contacts may be functional synapses, and they may be the morphological substrate of the galanin-controlled gonadal functions in humans.

Close anatomical associations between beta-endorphin and luteinizing hormone-releasing hormone neuronal systems in the human diencephalon.

Endogenous opiates, such as beta-endorphin, inhibit the release of luteinizing hormone (LH) release in the pituitary gland of several species including rat, pig, sheep, and human. Although it is generally believed that beta-endorphin influences gonadal functions via the regulation of hypothalamic LH-releasing hormone (LHRH) release, the morphological substrate underlying this regulation in humans remains elusive. In the present series of experiments the beta-endorphin-immunoreactive (IR) and LHRH-IR neural elements, utilizing single label immunohistochemistry, were mapped. Following the superimposition of the maps of these systems, the overlapping sites were identified and examined in order to verify the putative juxtapositions between the beta-endorphin-IR and LHRH-IR structures. LHRH-IR elements were detected mainly in the medial basal hypothalamus, in the medial preoptic area and along the diagonal band of Broca. Beta-endorphin-IR perikarya were observed in the infundibular region/median eminence, whereas beta-endorphin-IR axon varicosities were detected periventricularly in the preoptic and tuberal regions, in the medial basal hypothalamus and around the mamillary bodies. Careful examination of the immunoreactive elements in the overlapping areas revealed close contacts between beta-endorphin-IR and LHRH-IR structures, which have been verified in semithin plastic sections. These putative beta-endorphin-LHRH juxtapositions were most numerous in the medial preoptic area and in the infundibulum/median eminence of the human diencephalon. In conclusion, the present paper is the first study that revealed close juxtapositions between the beta-endorphin-IR and LHRH-IR neural elements in the human diencephalon. These beta-endorphin-LHRH contacts may be functional synapses, and they may be the morphological substrate of the beta-endorphin control on gonadal functions in man.

Estrogen prevents the loss of CA1 hippocampal neurons in gerbils after ischemic injury.

Estrogen replacement therapy is thought to attenuate the incidence of Alzheimer's disease in women and enhance cognitive functions. In rodents, estrogen protects cerebral cortical neurons from ischemic injury and cultured neurons from a variety of perturbations. Because few nuclear estrogen receptors have been detected in the dorsal hippocampus, the present studies used a global ischemia model to evaluate the neuroprotective actions of estrogen in this region. Ovariectomized gerbils were treated with placebo, 0.5 mg or 1 mg pellets of estradiol for 13 days. On day 7, the common carotid arteries were occluded for 5 min and on day 13 the animals were killed. Analysis of neurogranin mRNA, a marker of hippocampal neurons, with in situ hybridization revealed a dramatic and selective loss of CA1 neurons in the placebo-treated ovariectomized gerbils, whereas both 0.5 mg and 1 mg pellets of 17beta-estradiol prevented cell loss. Subsequent studies showed that a variety of estrogens, including diethylstilbestrol, estrone and 17alpha-estradiol as well as vitamin E, also protected CA1 neurons from ischemic injury in ovariectomized gerbils, whereas treatment with the estrogen antagonist tamoxifen was ineffective. The results of in vivo binding studies with 17alpha-iodovinyl-11beta-methoxyestradiol revealed a concentration of nuclear estrogen binding sites in the CA1 region of the ovariectomized gerbil brain, whereas binding in other hippocampal regions was limited. Moreover, the binding studies revealed that the regional pattern of binding was not altered after ischemic injury, with the exception of the hippocampus, where the binding sites were attenuated in ovariectomized animals with time after ischemic injury. Together, these data demonstrate that a variety of steroidal and non-steroidal estrogens are potent neuroprotective agents in an animal model of global ischemia, agents that protect neurons critical for learning and memory and susceptible to neurodegenerative diseases.

Catecholaminergic axons innervate LH-releasing hormone immunoreactive neurons of the human diencephalon.

Catecholamines have been shown to modulate gonadal functions via interactions with hypothalamic LH-releasing hormone (LHRH)-synthesizing neurons. To reveal the morphological background of this phenomenon, the distribution of LHRH neurons and tyrosine hydroxylase (TH)-immunoreactive (IR), catecholaminergic structures were mapped in the human diencephalon. First, the location of LHRH and TH-IR neuronal elements was analyzed, and then the relationship between the two different systems was examined. The LHRH-IR cell bodies were mainly present in the medial preoptic and infundibular areas. The TH-IR perikarya were located in the periventricular, paraventricular, and supraoptic hypothalamic nuclei and also in the median eminence. The TH-IR fibers were numerous in septal, infundibular, periventricular, and lateral hypothalamic regions. The brown, diaminobenzidine-labeled LHRH-containing perikarya were found to receive black, silver-intensified, TH-positive axon terminals in the infundibular and medial preoptic areas. However, in the preoptic and caudal parts of the diencephalon, only a few juxtapositions were noted. The present results indicate that hormone released from diencephalic LHRH-IR neurons in humans may be influenced by the central catecholaminergic system via direct synaptic mechanisms.

Estrogen receptor-beta immunoreactivity in luteinizing hormone-releasing hormone neurons of the rat brain.

Feedback regulation of luteinizing hormone-releasing hormone (LHRH) neurons by estradiol plays important roles in the neuroendocrine control of reproduction. Recently, we found that the majority of LHRH neurons in the rat contain estrogen receptor-beta (ER-beta) mRNA, whereas, they seemed to lack ER-alpha mRNA expression. In addition, we observed nuclear uptake of (125)I-estrogen by a subset of these cells. These data suggest that ER-beta is the chief receptor isoform mediating direct estrogen effects upon LHRH neurons. To verify the translation of ER-beta protein within LHRH cells, the present studies applied dual-label immunocytochemistry (ICC) to free-floating sections obtained from the preoptic area of rats. The improved ICC method using the silver-gold intensification of nickel-diaminobenzidine chromogen, enabled the observation of nuclear ER-beta-immunoreactivity in the majority of LHRH cells. The incidence of ER-beta expression was similarly high in LHRH neurons of ovariectomized female (87.8 +/- 2.3%, mean +/- SEM), estradiol-primed female (74.9 +/- 3.2%) and intact male (85.0 +/- 4.7%) rats. The presence of ER-beta mRNA, ER-beta immunoreactivity and (125)I-estrogen binding sites in LHRH neurons of the rat provide strong support for the notion that these cells are directly regulated by estradiol, through ER-beta. The gene targets and molecular mechanisms of this regulation remain unknown.

Estrogen receptor-alpha and beta- immunoreactivity and mRNA in neurons of sensory and autonomic ganglia and spinal cord.

Estrogen receptor-alpha immunoreactivity and mRNAs are present in neurons in locales that innervate genital organs, e.g., parasympathetic pelvic autonomic ganglia, sensory dorsal root and nodose ganglia, and autonomic areas of the lumbosacral spinal cord. With the availability of probes for the beta-isoform of the estrogen receptor, we studied this receptor in autonomic, sensory, and spinal cord neurons and compared it with the distribution of the alpha-receptor. Estrogen receptor-alpha and -beta immunoreactivity were located in the nuclei of neurons, were in subpopulations of parasympathetic neurons in pelvic ganglia, and sensory neurons of dorsal root and nodose ganglia. Both receptor subtypes were present in the lumbosacral spinal cord: in neurons of the outer laminae of the dorsal horn, lateral collateral and medial collateral pathways, sacral parasympathetic nucleus, dorsal intermediate gray, and lamina X. Similar numbers of spinal cord neurons were immunoreactive for estrogen receptor-beta and estrogen receptor-alpha. However, estrogen receptor-beta-immunoreactive neurons appeared less numerous in the outer dorsal horn, but more numerous in the deeper layers of the spinal cord than estrogen receptor-alpha neurons. Retrograde tracing from the uterus revealed "uterine-related" neurons in dorsal root and pelvic ganglia that contained estrogen receptor-alpha and -beta. In situ hybridization revealed both estrogen receptor-alpha and -beta mRNA transcripts in sensory neurons of the dorsal root and nodose ganglia, parasympathetic neurons of pelvic ganglia, and spinal cord neurons in the dorsal horn, sacral parasympathetic nucleus, and dorsal intermediate gray of L6-S1 segments. These studies show that both estrogen receptor-alpha and -beta are synthesized by autonomic and sensory neurons in parts of the nervous system that have connections with the female reproductive system. Such neurons contain neurotransmitters that have important functions in the female reproductive organs; thus, it is likely that estrogen can influence the activity of such neurons and consequently, through them, the activities of the reproductive organs.

Distribution of estrogen receptor beta immunoreactivity in the rat central nervous system.

The discovery of estrogen receptor beta (ER beta) and subsequent localization of its mRNA in the rat central nervous system (CNS) has provided new insights about estrogen action in brain. A critical step in understanding the role of ER beta is demonstrating that the mRNA is translated into functional protein. The present study used a new ER beta-specific polyclonal antiserum (Z8P) and immunocytochemistry (ICC) to investigate the distribution of ER beta in the rat CNS. Ovariectomized female rats were perfusion fixed, and free-floating sections were incubated with Z8P. After visualization with a standard ABC method, nuclear immunoreactivity was seen in neurons throughout the brain, including the olfactory nuclei, laminae IV-VI of the cerebral cortex, medial septum, preoptic area, bed nucleus of the stria terminalis, supraoptic nucleus, paraventricular nucleus, zona incerta, medial and cortical amygdaloid nuclei, cerebellum, nucleus of the solitary tract, ventral tegmental area, and spinal trigeminal nucleus. Moreover, the results of a double-label ICC/ in situ hybridization study revealed that ER beta mRNA and immunoreactivity were colocalized in neurons of the brain, thus confirming the specificity of the antiserum. Through the use of Western blot analysis, Z8P was shown to recognize in vitro translated ER beta, but not ER alpha, as well as a 60-kDa protein from rat granulosa cells and ovary extracts. The results of these studies have demonstrated that (1) ER beta mRNA is translated into immunoreactive protein throughout the rat brain, and (2) ER beta resides in the cell nucleus. Together, these data provide an anatomic foundation for future studies and advance our understanding of estrogen action in hypothalamic and extrahypothalamic brain regions.

Sex differences in progesterone receptor immunoreactivity in neonatal mouse brain depend on estrogen receptor alpha expression.

Around the time of birth, male rats express higher levels of progesterone receptors in the medial preoptic nucleus (MPN) than female rats, suggesting that the MPN may be differentially sensitive to maternal hormones in developing males and females. Preliminary evidence suggests that this sex difference depends on the activation of estrogen receptors around birth. To test whether estrogen receptor alpha (ER alpha) is involved, we compared progesterone receptor immunoreactivity (PRir) in the brains of male and female neonatal mice that lacked a functional ER alpha gene or were wild type for the disrupted gene. We demonstrate that males express much higher levels of PRir in the MPN and the ventromedial nucleus of the neonatal mouse brain than females, and that PRir expression is dependent on the expression of ER alpha in these regions. In contrast, PRir levels in neocortex are not altered by ER alpha gene disruption. The results of this study suggest that the induction of PR via ER alpha may render specific regions of the developing male brain more sensitive to progesterone than the developing female brain, and may thereby underlie sexual differentiation of these regions.

Estrogen receptor alpha, not beta, is a critical link in estradiol-mediated protection against brain injury.

Estradiol protects against brain injury, neurodegeneration, and cognitive decline. Our previous work demonstrates that physiological levels of estradiol protect against stroke injury and that this protection may be mediated through receptor-dependent alterations of gene expression. In this report, we tested the hypothesis that estrogen receptors play a pivotal role in mediating neuroprotective actions of estradiol and dissected the potential biological roles of each estrogen receptor (ER) subtype, ER alpha and ER beta, in the injured brain. To investigate and delineate these mechanisms, we used ER alpha-knockout (ER alpha KO) and ER beta-knockout (ER beta KO) mice in an animal model of stroke. We performed our studies by using a controlled endocrine paradigm, because endogenous levels of estradiol differ dramatically among ER alpha KO, ER beta KO, and wild-type mice. We ovariectomized ER alpha KO, ER beta KO, and the respective wild-type mice and implanted them with capsules filled with oil (vehicle) or a dose of 17 beta-estradiol that produces physiological hormone levels in serum. One week later, mice underwent ischemia. Our results demonstrate that deletion of ER alpha completely abolishes the protective actions of estradiol in all regions of the brain; whereas the ability of estradiol to protect against brain injury is totally preserved in the absence of ER beta. Thus, our results clearly establish that the ER alpha subtype is a critical mechanistic link in mediating the protective effects of physiological levels of estradiol in brain injury. Our discovery that ER alpha mediates protection of the brain carries far-reaching implications for the selective targeting of ERs in the treatment and prevention of neural dysfunction associated with normal aging or brain injury.

Topography and associations of luteinizing hormone-releasing hormone and neuropeptide Y-immunoreactive neuronal systems in the human diencephalon.

Neuropeptide Y (NPY) potentiates the effect of luteinizing hormone-releasing hormone (LHRH) on luteinizing hormone secretion in several species, including human. In addition to the pituitary sites, the interactions of the NPY and LHRH systems may involve diencephalic loci. However, the morphologic basis of this putative communication has not yet been elucidated in the human brain. To discover interaction sites, the distribution and connections of LHRH and NPY-immunoreactive (IR) neuronal elements in the human hypothalamus were investigated by means of light microscopic single- and double-label immunocytochemistry. NPY-IR perikarya and fibers were found to be widely distributed in the ventral diencephalon, with high densities in the preopticoseptal, periventricular, and tuberal regions. Small neuronal cell groups were infiltrated with a dense network of varicose NPY-IR fibers in the lateral preoptic area. The LHRH-IR perikarya were located mainly in the preopticoseptal region, diagonal band of Broca, lamina terminalis, and periventricular and infundibular nuclei. A few LHRH-IR neurons and fibers were scattered in the mamillary region. The overlap between the NPY and LHRH systems was apparent in the periventricular, paraventricular, and infundibular nuclei. Double-labeling immunohistochemistry showed NPY-IR axon varicosities in contact with LHRH-IR perikarya and main dendrites. The putative innervation of LHRH neurons by NPY-IR fibers was also seen in 1-microm-thick plastic sections and with confocal laser scanning microscope, thus further supporting the functional impact of NPY-IR terminals on LHRH-IR neurons. The present findings suggest that the hypophysiotropic LHRH-synthesizing neurons may be innervated by intrahypothalamic NPY-IR fibers. Confirmation by ultrastructural analysis would demonstrate that the LHRH system in the human hypothalamus is regulated by NPY, as has been demonstrated in nonhuman species.

Evidence for novel estrogen binding sites in the rat hippocampus.

Estrogen modulates the morphology and physiology of the rat hippocampus and enhances cognitive function. While estrogen receptor (alpha and beta) messenger RNAs have been detected in the hippocampus, the presence of functional protein remains uncertain. The present study used a new radiolabeled estrogen, [125I]estrogen, and in vivo autoradiography to address this question. Nuclear uptake and retention of [125I]estrogen was detected in the pyramidal cells of CA1-CA3, with the majority of cells in the ventral horn of CA2 and CA3 being labeled. Additional labeled cells were scattered throughout the strata oriens and radiatum and the hilus of the dentate gyrus. Since the number and distribution of labeled cells in the hippocampus was more than expected, in situ hybridization was used to assess the localization of estrogen receptor (alpha and beta) messenger RNAs in this brain region. The results revealed that both estrogen receptors are expressed in regions where [125I]estrogen binding was seen, although the intensity of estrogen receptor-alpha hybridization signal appears to be stronger when compared with estrogen receptor-beta.The results of these studies have demonstrated the presence of estrogen receptors in rat hippocampus and shown that the distribution of binding sites was much greater than expected, particularly in the pyramidal cells of the ventral hippocampus. These observations challenge our current thinking about steroid hormones and their mechanism(s) of action in a region associated with learning and memory and affected by the neurodegenerative conditions of aging.

Detection of estrogen receptor-beta messenger ribonucleic acid and 125I-estrogen binding sites in luteinizing hormone-releasing hormone neurons of the rat brain.

Luteinizing hormone-releasing hormone (LHRH) neurons of the forebrain play a pivotal role in the neuroendocrine control of reproduction. Although serum estrogen levels influence many aspects of LHRH neuronal activity in the female, earlier studies were unable to detect estrogen receptors (ERs) within LHRH neurons, thus shaping a consensus view that the effects of estradiol on the LHRH neuronal system are mediated by interneurons and/or the glial matrix. The present studies used dual-label in situ hybridization histochemistry (ISHH) and combined LHRH-immunocytochemistry/125I-estrogen binding to readdress the estrogen-receptivity of LHRH neurons in the female rat. In ISHH experiments we found that the majority of LHRH neurons exhibited hybridization signal for the "beta" form of ER (ER-beta). The degree of colocalization was similar in topographically distinct populations of LHRH neurons and was not significantly altered by estradiol (67.2+/-1.8% in ovariectomized and 73.8+/-4.2% in ovariectomized and estradiol-treated rats). In contrast, the mRNA encoding the classical ER-alpha could not be detected within LHRH neurons. In addition, in vivo binding studies using 125I-estrogen revealed a subset of LHRH-immunoreactive neurons (8.8%) which accumulated the radioligand thus providing evidence for the translation of ER protein(s) within these cells. The findings that most LHRH neurons in the female rat express ER-beta mRNA and at least some are capable of binding 125I-estrogen challenge the current opinion that estrogen does not exert direct effects upon the LHRH neuronal system.

Estrogen binding and estrogen receptor characterization (ERalpha and ERbeta) in the cholinergic neurons of the rat basal forebrain.

Estrogen is thought to enhance cognitive functions by modulating the production of acetylcholine in basal forebrain neurons; a system that projects to the cerebral cortex and hippocampus and plays a central role in learning and memory. To elucidate the mechanism of estrogen action in the cholinergic system, we utilized a combined in vivo autoradiography/immunocytochemistry technique to evaluate the distribution of estrogen binding sites in cholinergic neurons of the rat basal forebrain. The results of these studies revealed that a portion of the cholinergic neurons in the medial septum (41%), vertical (32%) and horizontal (29%) limbs of the diagonal band and in the substantia innominata/nucleus basalis (4%) contained estrogen receptors. Through the use of a double-label in situ hybridization/immunocytochemistry technique we have shown that estrogen receptor-alpha is the predominant estrogen receptor in the cholinergic neurons, with only a few cells containing estrogen receptor-beta. The results of these studies provide evidence that biologically active estrogen receptors are present in the basal forebrain cholinergic neurons of the adult rat brain, with estrogen receptor-alpha being the predominant receptor subtype. The demonstration that cholinergic neurons contain estrogen receptors is consistent with the possibility that estrogen directly modulates the activity of cholinergic neurons in rats and may provide insight as to how estrogen improves cognitive functions in women.

Estrogen is more than just a "sex hormone": novel sites for estrogen action in the hippocampus and cerebral cortex.

For decades estrogen was thought of only as a "sex hormone," as it plays a fundamental role in regulating behavioral and physiological events essential for successful procreation. In recent years, estrogen has been shown to exert effects on the structure and function of the hippocampus and cortex. The discovery of a new estrogen receptor (ER-beta) and localization of ER-alpha and ER-beta mRNAs in the pyramidal cells of the rat hippocampus and ER-beta mRNA in rat cortex have provided new insight into how estrogen may directly modulate the structure and function of these neurons. Moreover, recent in vivo (125)I-estrogen binding studies have shown that nuclear estrogen binding sites are widely distributed in the pyramidal cells throughout CA1-3 of the hippocampus and laminae II-VI of the isocortex, demonstrating that ER mRNAs are translated into biologically active protein. The functional impact of estrogen receptor localization in the cortex and hippocampus may prove relevant to the emerging role for estrogen as a protective factor in neurodegenerative injury. This potential role is further highlighted by the recent findings that the expression of ER-alpha and ER-beta changes following ischemic brain injury and that these changes correlate with the hormonal modulation of protective factors. These data provide the first evidence that the expression of ERs in the adult cortex is not static, but instead, responsive to neuronal injury and perhaps additional factors that influence the cortical environment and status of these neurons. Together, these data indicate that estrogen has a far greater effect on the hippocampus and isocortex than previously thought. Furthermore, these new findings challenge our current thinking about steroid hormones and their mechanism(s) of action in regions associated with learning and memory and affected by the neurodegenerative conditions of aging.

Estrogen receptor beta and progesterone receptor mRNA in the intergeniculate leaflet of the female rat.

The present study was undertaken to explore the possibility that the integration of hormonal cues in the regulation of neuroendocrine mechanisms may occur outside of the hypothalamus at the level of the lateral geniculate body. In situ hybridization for mRNA encoding estrogen receptor beta and progesterone receptor was carried out on sections containing the lateral geniculate body using [35S]-labeled antisense riboprobes. Labeled cells were present in different limbic and hypothalamic sites as described previously. Populations of cells distributed homogeneously in the ventral lateral geniculate nucleus and intergeniculate leaflet were also found to express mRNA for estrogen receptor beta and progesterone receptor. The dorsal lateral geniculate nucleus lacked specific labeling for either type of gonadal steroid hormone receptor mRNA. The present observation together with the recent demonstration of a direct pathway between the intergeniculate leaflet and hypothalamic neuroendocrine cells indicate that integration of hormonal and photic stimuli in the central regulation of endocrine mechanisms occurs outside of the hypothalamus in the lateral geniculate body.

Estrogen receptor immunoreactivity is present in the majority of central histaminergic neurons: evidence for a new neuroendocrine pathway associated with luteinizing hormone-releasing hormone-synthesizing neurons in rats and humans.

The central regulation of the preovulatory LH surge requires a complex sequence of interactions between neuronal systems that impinge on LH-releasing hormone (LHRH)-synthesizing neurons. The reported absence of estrogen receptors (ERs) in LHRH neurons indicates that estrogen-receptive neurons that are afferent to LHRH neurons are involved in mediating the effects of this steroid. We now present evidence indicating that central histaminergic neurons, exclusively located in the tuberomammillary complex of the caudal diencephalon, serve as an important relay in this system. Evaluation of this system revealed that 76% of histamine-synthesising neurons display ERalpha-immunoreactivity in their nucleus; furthermore histaminergic axons exhibit axo-dendritic and axo-somatic appositions onto LHRH neurons in both the rodent and the human brain. Our in vivo studies show that the intracerebroventricular administration of the histamine-1 (H1) receptor antagonist, mepyramine, but not the H2 receptor antagonist, ranitidine, can block the LH surge in ovariectomized estrogen-treated rats. These data are consistent with the hypothesis that the positive feedback effect of estrogen in the induction of the LH surge involves estrogen-receptive histamine-containing neurons in the tuberomammillary nucleus that relay the steroid signal to LHRH neurons via H1 receptors.