Anti-ADDL antibodies differentially block oligomer binding to hippocampal neurons.

Abeta-derived diffusible ligands (ADDLs) are abundant in AD brain, bind to hippocampal neurons and induce deficits in rodent cognition. To further investigate ADDL binding to neurons and identify antibodies that block this association, a panel of anti-Abeta and anti-ADDL antibodies was characterized for their ability to immuno-detect neuronally bound ADDLs and attenuate the binding of ADDLs to neurons. The results showed that anti-Abeta and anti-ADDL antibodies were able to abate ADDLs binding to hippocampal neurons, but to different degrees. Quantitative assessment of binding showed that one antibody, ACU-954 was markedly more effective at blocking ADDL binding than other antibodies assessed. ACU-954 was also found to block ADDL binding to hippocampal slice cultures, attenuate the ADDL-induced loss of dendritic spines and detect "natural ADDLs" in human AD tissue. These results demonstrated that antibodies that bind to and block ADDL binding to neurons can be identified, although their efficacy is conformationally specific since it is not readily apparent or predictable based on the core linear epitope or affinity for monomeric Abeta.

Expression of Disrupted-In-Schizophrenia-1, a schizophrenia-associated gene, is prominent in the mouse hippocampus throughout brain development.

DISC1 (Disrupted-In-Schizophrenia 1) has been associated with schizophrenia in multiple genetic studies. Studies from our laboratory have shown that Disc1, the mouse ortholog of DISC1, is highly expressed in the dentate gyrus of the hippocampus in the adult mouse brain. Because developmental dysfunction of the hippocampus is thought to play a major role in schizophrenia pathogenesis, and the dentate gyrus is a major locus for adult neurogenesis in the mouse, we investigated Disc1 expression during mouse brain development. Strikingly, Disc1 is strongly expressed in the hippocampus during all stages of hippocampal development, from embryonic day 14 through adulthood. Disc1 mRNA was detected in the dentate gyrus at all stages in which this structure was identifiable, as well as in the cornu ammonis (CA) fields of the hippocampus, the subiculum and adjacent entorhinal cortex, and the developing cerebral neocortex, hypothalamus, and olfactory bulbs, all of which also express Disc1 in the adult mouse brain. In addition, Disc1 mRNA was seen in regions of the developing mouse brain which do not express Disc1 during adulthood, regions including the bed nucleus of the stria terminalis, reticular thalamic nucleus and reuniens thalamic nucleus. These results demonstrate that Disc1 marks the hippocampus from its earliest stages, and suggest that developmental Disc1 dysfunction may lead to defects in hippocampal function that are associated with schizophrenia.

Sex-related differences in the distribution of opioid receptor-like 1 receptor mRNA and colocalization with estrogen receptor mRNA in neurons of the spinal trigeminal nucleus caudalis in the rat.

We recently reported that exogenously applied orphanin FQ, the endogenous ligand for opioid receptor-like 1 (ORL(1)) receptor, produces sex-specific modulation of trigeminal nociception, and that estrogen contributes to these sex-related differences. Estrogen could produce these sex-related differences by altering the expression of the ORL(1)-receptor gene in the trigeminal nucleus caudalis. Utilizing in situ hybridization, we compared levels of ORL(1) receptor mRNA and investigated its colocalization with estrogen receptor mRNA in trigeminal neurons. Our results showed that in male rats, ORL(1) receptor mRNA is abundantly expressed in the rostral part of the trigeminal nucleus caudalis, and at the junction of caudalis and interpolaris (Vc/Vi). In comparison with males, levels of ORL(1) receptor mRNA were not significantly different in proestrus females, but were significantly higher in the rostral trigeminal nucleus caudalis and at the junction of Vc/Vi of diestrus females. In addition, ovariectomy raised the levels in the rostral trigeminal nucleus caudalis, and at the junction of Vc/Vi. Levels were reduced to proestrus levels in these regions following estradiol replacement. Our results also showed that ORL(1) receptor mRNA is present in majority of estrogen receptor (alpha and/or beta) mRNA-containing neurons. We conclude that there are sex-related differences in the ORL(1)-receptor gene expression in the trigeminal nucleus caudalis, which appear to be determined in part by estrogen levels.

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.

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.

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, 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.

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 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 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 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.

Estradiol modulates bcl-2 in cerebral ischemia: a potential role for estrogen receptors.

We have shown that physiological levels of estradiol exert profound protective effects on the cerebral cortex in ischemia induced by permanent middle cerebral artery occlusion. The major goal of this study was to begin to elucidate potential mechanisms of estradiol action in injury. Bcl-2 is a proto-oncogene that promotes cell survival in a variety of tissues including the brain. Because estradiol is known to promote cell survival via Bcl-2 in non-neural tissues, we tested the hypothesis that estradiol decreases cell death by influencing bcl-2 expression in ischemic brain injury. Furthermore, because estradiol may protect the brain through estrogen receptor-mediated mechanisms, we examined expression of both receptor subtypes ERalpha and ERbeta in the normal and injured brain. We analyzed gene expression by RT-PCR in microdissected regions of the cerebral cortex obtained from injured and sham female rats treated with estradiol or oil. We found that estradiol prevented the injury-induced downregulation of bcl-2 expression. This effect was specific to bcl-2, as expression of other members of the bcl-2 family (bax, bcl-x(L), bcl-x(S), and bad) was unaffected by estradiol treatment. We also found that estrogen receptors were differentially modulated in injury, with ERbeta expression paralleling bcl-2 expression. Finally, we provide the first evidence of functional ERbeta protein that is capable of binding ligand within the region of the cortex where estradiol-mediated neuroprotection was observed in cerebral ischemia. These findings indicate that estradiol modulates the expression of bcl-2 in ischemic injury. Furthermore, our data suggest that estrogen receptors may be involved in hormone-mediated neuroprotection.

Biologically active estrogen receptor-beta: evidence from in vivo autoradiographic studies with estrogen receptor alpha-knockout mice.

Estrogen receptor-1 (ER beta) messenger RNA (mRNA) has been detected in the brain of wild-type and estrogen receptor-alpha knockout (ER alphaKO) mice. The present study used in vivo autoradiography to evaluate the binding of 125I-estrogen, a compound with a similar affinity for both ERs to ascertain whether ER beta mRNA is translated into biologically active receptor. Mice were injected with 125I-estrogen, and sections were mounted on slides and opposed to emulsion. After exposure, labeled cells were seen in ER alphaKO brain regions where ER beta is expressed (preoptic and paraventricular nuclei of the hypothalamus; bed nucleus of the stria terminalis; amygdala; entorhinal cortex; and dorsal raphe). Competition studies with 17beta-estradiol eliminated binding in the ER alphaKO brain, whereas 16alphaIE2, an ER alpha selective agonist and dihydrotestosterone had no effect. In contrast, competition studies with 16alphaIE2 in wild-type mice eliminated 125I-estrogen binding to ER alpha and resulted in a pattern of residual binding comparable to that seen in the ER alphaKO brain. The results demonstrate that residual estrogen binding sites are present in regions of the ER alphaKO brain where ER beta is expressed, brain regions that were also seen after eliminating binding to ER alpha in wild-type mice. These data provide the first evidence that ER beta mRNA is translated into a biologically active protein in the rodent brain.

The effects of progesterone on oxytocin mRNA levels in the paraventricular nucleus of the female rat can be altered by the administration of diazepam or RU486.

Oxytocin (OT) facilitates the onset of maternal behaviour in the late pregnant rat, enhances uterine contractility at parturition, and elicits milk ejection during lactation. If the rising estradiol (E2 and declining progesterone (P) of late pregnancy is reproduced in a virgin ovariectomized rat by implanting E2- and P-filled capsules for 2 weeks followed by removal of P-containing implants 36-48 h prior to death, OT messenger ribonucleic acid (mRNA) levels increase in the paraventricular and supraoptic nuclei (PVN and SON) of the rat. Both E2 administration and P withdrawal are necessary to increase OT mRNA, but the mechanisms of these effects are not understood. P may work within the PVN although P receptors are reported to be sparse or non-existent in the PVN or outside the PVN on PR-containing neurones that project to OT-containing neurones or via membrane bound receptors that are known to bind neurosteroids and gamma aminobutyric acid (GABA). To determine the mechanism through which P may inhibit or P withdrawal may increase OT mRNA levels, virgin ovariectomized (OVX) rats received sequential E2 and P via Silastic implants for 14 days. On day 13, prior to removal of P capsules on day 14, the rats were given the benzodiazepine agonist, diazepam, or saline injections subcutaneously (s.c.) twice daily until death on day 16. OT mRNA levels were increased in the steroid-treated group that received saline but not diazepam. In experiment 2, P capsules were removed on day 14 or pharmacological P withdrawal was induced by injecting RU486 injections s.c. twice daily until death 48 h later. OT mRNA levels were increased in the steroid-treated group that received RU486. Subsequent studies demonstrated the expression of PR mRNA within the rat PVN. The data suggest that gonadal steroids may influence PVN OT mRNA levels by modulating the GABA(A) receptor or by directly altering gene transcription via the PR.

Distribution of pre-pro-glucagon and glucagon-like peptide-1 receptor messenger RNAs in the rat central nervous system.

Glucagon-like peptide-1 (GLP-1) is derived from the peptide precursor pre-pro-glucagon (PPG) by enzymatic cleavage and acts via its receptor, glucagon-like peptide-1 receptor (GLP-1R). By using riboprobes complementary to PPG and GLP-1R, we described the distribution of PPG and GLP-1R messenger RNAs (mRNAs) in the central nervous system of the rat. PPG mRNA-expressing perikarya were restricted to the nucleus of the solitary tact or to the dorsal and ventral medulla and olfactory bulb. GLP-1R mRNA was detected in numerous brain regions, including the mitral cell layer of the olfactory bulb; temporal cortex; caudal hippocampus; lateral septum; amygdala; nucleus accumbens; ventral pallium; nucleus basalis Meynert; bed nucleus of the stria terminalis; preoptic area; paraventricular, supraoptic, arcuate, and dorsomedial nuclei of the hypothalamus; lateral habenula; zona incerta; substantia innominata; posterior thalamic nuclei; ventral tegmental area; dorsal tegmental, posterodorsal tegmental, and interpeduncular nuclei; substantia nigra, central gray; raphe nuclei; parabrachial nuclei; locus ceruleus, nucleus of the solitary tract; area postrema; dorsal nucleus of the vagus; lateral reticular nucleus; and spinal cord. These studies, in addition to describing the sites of GLP-1 and GLP-1R synthesis, suggest that the efferent connections from the nucleus of the solitary tract are more widespread than previously reported. Although the current role of GLP-1 in regulating neuronal physiology is not known, these studies provide detailed information about the sites of GLP-1 synthesis and potential sites of action, an important first step in evaluating the function of GLP-1 in the brain. The widespread distribution of GLP-1R mRNA-containing cells strongly suggests that GLP-1 not only functions as a satiety factor but also acts as a neurotransmitter or neuromodulator in anatomically and functionally distinct areas of the central nervous system.

Evidence for the colocalization of estrogen receptor-beta mRNA and estrogen receptor-alpha immunoreactivity in neurons of the rat forebrain.

Estrogen receptor beta (ER beta) mRNA is expressed in several rat brain regions where ER alpha is abundant. In vitro studies have shown that ER alpha and ER beta can heterodimerize and that the activity of this complex may be different than an ER alpha or ER beta homodimer complex. The purpose of the present study was to ascertain if ER alpha and ER beta are co-expressed by certain neuronal populations using a double label in situ hybridization/immunocytochemistry method. The results revealed that neurons in the bed nucleus of the stria terminalis, medial amygdala and preoptic area contain both ERs, with the vast majority of the neurons being double labeled. In other brain regions including the arcuate nucleus, cortical amygdaloid nuclei and ventromedial nucleus, only a few double-labeled cells were detected, while neurons in the paraventricular nucleus, supraoptic nucleus, and cerebral cortex expressed only ER beta mRNA. The results of these double label experiments provide the first evidence that ER alpha and ER beta coexist in neurons under in vivo conditions and suggest that estrogens may differentially modulate the activity of certain neuronal populations depending on whether the cells expresses ER alpha, ER beta or both ERs.

The estrogen receptor beta subtype: a novel mediator of estrogen action in neuroendocrine systems.

The recent discovery that an additional estrogen receptor (ERbeta) subtype is present in many rat, mouse, and human tissues has advanced our understanding of the mechanisms underlying estrogen signalling. Ligand-binding experiments have shown specific binding of 17beta-estradiol by ERbeta with an affinity similar to that of ERalpha. The rat tissue distribution and/or the relative level of ERalpha and ERbeta expression seems to be quite different, i.e., moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ERalpha and prostate, ovary, lung, bladder, brain, bone, uterus, and testis for ERbeta. Within the same organ it often appears that the ER subtypes are expressed in different cell types, supporting the hypothesis that the ER's may have different biological functions. The cell type-specific expression of ERalpha and ERbeta in rat prostate, testis, uterus, ovary, and brain and the distribution of ERbeta mRNA in the ERalpha knock-out mouse brain are discussed. The discovery of ERbeta suggests the existence of two previously unrecognized pathways of estrogen signalling; via the ERbeta subtype in tissues exclusively expressing this subtype and via the formation of heterodimers in tissues expressing both ER subtypes. The existence of two ER subtypes, their differential expression pattern, and different actions on certain response elements could provide explanations for the striking species-, cell-, and promoter-specific actions of estrogens and antiestrogens. The challenge for the future is to unravel the detailed physiological role of each subtype and to use this knowledge to develop the next generation of ER-targeted drugs with improved therapeutic profiles in the treatment or prevention of osteoporosis, cardiovascular system disorders, Alzheimer's disease, breast cancer, and disorders of the urogenital tract.