Estrogen and androgen differentially modulate the growth-associated protein GAP-43 (neuromodulin) messenger ribonucleic acid in postnatal rat brain.

The level of expression of the gene encoding GAP-43, a protein implicated in neurite outgrowth and motility, is sexually dimorphic in the postnatal brain and modulated by testosterone (T). To determine which metabolite of T modulates the expression of GAP-43 in the postnatal brain, the present study investigated the effect of estrogen and androgen receptor antagonists, tamoxifen (TAM) and cyproterone acetate (CA), and agonists, diethylstilbestrol (DES) and dihydrotestosterone (DHT). On postnatal day 1, male rats were injected sc daily with oil, TAM, or CA, whereas additional female pups were treated with oil, DES, or DHT. On postnatal day 6, brains were collected and 16-micron cryostat sections processed and hybridized with a 35S-labeled antisense riboprobe complementary to GAP-43 messenger RNA (mRNA). A comparison of hybridization signal in the cerebral cortex, bed nucleus of the stria terminalis, and medial preoptic nucleus demonstrated that the level of GAP-43 mRNA in males was significantly higher than in females. However, when male pups were injected with CA, a female-like level of GAP-43 mRNA was measured in the cerebral cortex. The low level of GAP-43 hybridization signal in the female cortex was markedly increased when females were treated with DHT. In contrast, the level of GAP-43 mRNA in the male medial preoptic nucleus was attenuated after treatment with TAM, whereas the level in the female was augmented with DES. Interestingly, the level of GAP-43 mRNA in the bed nucleus was reduced when males were treated with either TAM or CA and augmented when females were administered either DES or DHT. The results of these studies indicate that the effects of T on GAP-43 mRNA levels in the postnatal brain are modulated by estrogen- and androgen receptor-mediated events in a region-specific manner.

Glucagon-like peptide-1 receptor (GLP1-R) mRNA in the rat hypothalamus.

GLP-1 has been shown to dramatically reduce food intake in fasted rats and is thought to exert its effects by modulating neuronal function in the hypothalamus. To date, little is known about the distribution of GLP1-R and its mRNA in the rodent hypothalamus. The purpose of the present study was to utilize in situ hybridization histochemistry to determine the anatomical distribution of GLP1-R mRNA in the rat hypothalamus. The results of these studies revealed an extensive distribution of GLP1-R mRNA throughout the rostral-caudal extent of the hypothalamus; with a dense accumulation of labeled cells in the supraoptic, paraventricular, and arcuate nuclei. Additional labeled cells were also detected in medial and lateral preoptic areas, periventricular nucleus, ventral division of the bed nucleus of the stria terminalis, lateral hypothalamus, and dorsomedial nucleus. The results of these in situ hybridization histochemical studies have provided detailed and novel information about the distribution of GLP1-R mRNA in the rat hypothalamus. In addition, this morphological data provides important information about the neuronal systems modulated by GLP-1 and their potential role in feeding behavior.

Regulation of progesterone receptor messenger ribonucleic acid in the rat medial preoptic nucleus by estrogenic and antiestrogenic compounds: an in situ hybridization study.

Progesterone receptor (PR) messenger RNA (mRNA) is concentrated in neurons of the preoptic area and other regions of the rat hypothalamus where it is colocalized with the estrogen receptor and regulated by changes in the steroid hormonal milieu. To date, little is known about the regulation of PR mRNA by estrogens and whether antiestrogenic compounds are capable of modulating its expression. The present studies used in situ hybridization to ascertain the time course of PR mRNA regulation in the medial preoptic nucleus by 17beta-estradiol, determine the effective dose required to elicit a response, and compare the efficacy of 17beta-estradiol with a variety of estrogenic or antiestrogenic compounds. The first series of studies revealed that the treatment of ovariectomized rats with 17beta-estradiol resulted in an increase in PR expression within 2 h, after which it remained elevated until 10 h postinjection and then returned to baseline levels. When ovariectomized rats were injected with 25-1000 ng/kg of 17beta-estradiol and euthanized 6 h later, a dose-dependent increase in the level of PR mRNA was observed, with a maximal response at 1000 ng/kg and an EC50 of 93.5 ng/kg. Subsequent studies evaluated the efficacy of a variety of estrogenic and antiestrogenic compounds in the rat preoptic nucleus. 17Beta-estradiol, diethylstilbestrol, and 17alpha-estradiol all significantly increased the level of PR mRNA, although the degree of induction varied with each compound. The injection of tamoxifen, raloxifene, toremifene, droloxifene, clomiphene, GW 5638, or ICI 182,780 had no significant estrogenic effect on PR gene expression at the dose evaluated. In contrast, when tamoxifen or raloxifene, but not ICI 182,780, was administered in the antagonist mode, a significant dose-related decrease in the estradiol-induced level of PR mRNA was seen in the preoptic area. The results of these studies clearly demonstrate that PR mRNA expression in the rat preoptic area is rapidly stimulated by a small dose of 17beta-estradiol. Moreover, the present report has also shown that the estrogenic nature of compounds such as tamoxifen, raloxifene, toremifene, droloxifene, clomiphene, and GW 5638 cannot be predicted by their activity in peripheral tissues. Together, the results of these studies provide important information about the central activity of estrogens and provide evidence for their tissue-specifc actions in the rat.

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.

Progestin target cell distribution in forebrain and midbrain regions of the 8-day postnatal mouse brain.

The present study investigated the anatomical distribution of progestin target cells throughout the forebrain and midbrain regions of the 8-day postnatal female mouse. Female ICR mice were sc injected with 100 micrograms/100 g BW estradiol valerate on postnatal day 5 (birth = day 0). On postnatal day 8, treated mice were sc injected with 0.32 micrograms/100 g BW (Z)-17 beta-hydroxy-17 alpha-(2-[125I]iodovinyl)4-estren-3-one ([125I] progestin). For competition, additional estrogen-treated mice were each injected with 320 micrograms R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione; a potent synthetic progestin), 320 micrograms dihydrotestosterone, or 32, 160, or 320 micrograms corticosterone 1 h before [125I]progestin to show the specificity of [125I]progestin for the progestin receptor. Two hours after injection of [125I]progestin, the brains were removed, frozen, and processed for high resolution thaw-mount autoradiography. After 8-60 days of exposure, nuclear uptake and retention of [125I]progestin were detected in many brain regions, including the septum; bed nucleus of the stria terminalis; and preoptic area, periventricular nucleus, ventromedial nucleus, arcuate nucleus, and dorsomedial nucleus of the hypothalamus. In addition, labeling was seen in the cerebral cortex, caudate putamen, hippocampus, amygdala, and substantia nigra. Competition studies showed that excess R5020 prevented nuclear concentration of ligand, while dihydrotestosterone and corticosterone did not. The results indicate that the distribution of progestin target cells in extrahypothalamic regions of the developing brain is more extensive than that in the adult, while a similar topography was seen in the preoptic area and hypothalamus. The results further suggest that progestin action during brain development may influence the growth and development of target cells not only in the hypothalamus but also in regions of the brain previously not considered to be sites of hormone action.

The distribution of progesterone receptor in the 20-day-old fetal mouse: an autoradiographic study with [125I]progestin.

The distribution of progestin target sites in 20-day-old fetuses of estrogen-primed pregnant mice was investigated by thaw-mount autoradiography. Pregnant mice received a Silastic estradiol implant on day 17 and were ovariectomized on day 19 of pregnancy. Twenty-four hours after ovariectomy 10 prematurely delivered fetuses were each injected with 0.33 microgram/100 g BW [125I]progestin (SA, 2200 Ci/mM). To show specificity of progestin localization two additional fetuses were each injected sc with 20 micrograms R5020, a synthetic progestin, 1 h before the injection of [125I]progestin. The fetuses were frozen 2 h after injection of [125I]progestin, sectioned, and processed for thaw-mount autoradiography. Cells with nuclear uptake and retention of radioactivity were observed in numerous tissues, including certain regions of the oral mucosa and developing teeth, esophagus, larynx, skin, mammary gland, skeletal muscle, kidney, and reproductive glands and ducts. Injection of unlabeled R5020 1 h before [125I]progestin prevented nuclear concentration of radioactivity in all target tissues. The results indicate that progesterone receptors are expressed with a regional, cellular, and subcellular distribution in term fetal mouse tissues and suggest that progesterone is important to the growth and development of certain fetal tissues.

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.

Estrogen receptor messenger ribonucleic acid in female rat brain during the estrous cycle: a comparison with ovariectomized females and intact males.

Variations in levels of estrogen receptor mRNA were investigated in the medial preoptic nucleus, arcuate nucleus, and ventromedial nucleus of the hypothalamus throughout the phases of the female estrous cycle and compared with those in ovariectomized female and intact male rats. Female Wistar rats were killed during estrus, metestrus, diestrus, or proestrus or 72 h after ovariectomy as were a group of intact male rats. Brains were removed and frozen, and 20-microns cryostat sections were thaw-mounted onto slides and hybridized with a 35S-labeled antisense estrogen receptor probe. Section-mounted slides were processed, apposed to x-ray film, then dipped in liquid emulsion, and quantified. After exposure, estrogen receptor mRNA was detected in several brain regions, including the medial preoptic nucleus, arcuate nucleus, and ventromedial nucleus of the hypothalamus. Estrogen receptor mRNA levels in the medial preoptic nucleus were highest during estrus and metestrus, attenuated at diestrus, and low during proestrus. In contrast, the hybridization signal in the arcuate and ventromedial nuclei was low during estrus and then gradually increased throughout the cycle until it peaked during proestrus. Ovariectomized females exhibited an elevated level of estrogen receptor mRNA in all brain regions investigated. Hybridization signal in male medial preoptic nucleus and ventromedial nucleus was reduced compared with those in both intact and ovariectomized females. Estrogen receptor mRNA levels in the arcuate nucleus were similar to those in intact females, but less than those in ovariectomized animals. The results of these studies demonstrate that estrogen receptor mRNA levels are sexually dimorphic, vary during the estrous cycle, and increase after ovariectomy. Furthermore, these results indicate that the magnitude and direction of change observed during the estrous cycle are region specific and suggest that factors other than endogenous estrogen levels differentially modulate estrogen receptor mRNA expression in the hypothalamus.

Progestin receptors in brain and pituitary of 20-day-old fetal mice: an autoradiographic study using [125I]progestin.

The distribution of progestin target sites in the brain and pituitary of estrogen-primed 20-day-old fetal mice was investigated by thaw-mount autoradiography. Three pregnant mice were each implanted sc with a Silastic tube containing estrogen on day 17 and ovariectomized on day 19 of gestation. Twenty-four hours after ovariectomy 10 fetuses (5 males and 5 females) were collected and each injected sc with 0.33 microgram/100 g BW [125I]progestin (SA, 2200 Ci/mM). For competition, two additional fetuses were injected with 20 micrograms R5020 1 h before (Z)-17 beta-hydroxy-17 alpha-(2[125I]iodovinyl)4-estren-3-one [( 125I]Progestin) to demonstrate that nuclear uptake and retention of radioactivity were specific for progestin. Two hours after injection of [125I]Progestin all fetuses were mounted, frozen, and sectioned in a cryostat. After 1-37 days of exposure, sections were developed and scanned for labeled cells. Cells with nuclear concentration were found in the male and female preoptic area, within certain nuclear groups in the basal hypothalamus, in the central gray of the midbrain, and in the pituitary. No labeling was detected in the cortex or amygdala. The results indicate that cells in certain regions of the brain and pituitary express progestin receptors at the end of gestation and suggest that progesterone is important for the normal development of these cells.

Developmental changes in estrogen receptors in mouse cerebral cortex between birth and postweaning: studied by autoradiography with 11 beta-methoxy-16 alpha-[125I]iodoestradiol.

The presence of estrogen receptor cells in postnatal cerebral cortex and their topographical and numerical changes between birth and postweaning were examined. On postnatal days 0, 2, 8, 12, 18, and 25, six mice (three males and three females) were injected sc with 0.25 microgram/100 g BW 11 beta-methoxy-16 alpha-[125I]iodoestradiol [( 125I]MIE2). Two additional males on postnatal day 2 were each sc injected with 250 micrograms/100 g BW 17 beta-estradiol 1 h before radiolabeled ligand to establish the specificity of nuclear label. Two hours after the injection of [125I]MIE2 brains were frozen, and 4-microns sections were thaw-mounted and processed for autoradiography. Autoradiograms were exposed for 1-45 days, and cortical cells with nuclear uptake and retention of [125I]MIE2 were evaluated at the levels of the frontal pole, preoptic area, and central and posterior hypothalamus. At birth, cells with nuclear label were found predominantly in deep cortical layers. Between birth and postnatal day 2, the number of labeled cells increased in deep and intermediate laminae and first appeared in certain superficial regions. By day 8, labeled cells were concentrated in laminae II-VI of the cingulate/paracingulate and suprarhinal cortex. On day 12, labeling in laminae V and VI declined to a few cells, while a concentration of labeled cells remained in laminae II and III of the cingulate/paracingulate and suprarhinal regions. With subsequent development, an attenuation in labeling at all cortical levels was observed. By day 25, a small cluster of labeled cells remained in lamina II and III of the anterior cingulate, paracingulate, and suprarhinal regions, with additional labeled cells scattered throughout the remaining cortex. Competition with unlabeled 17 beta-estradiol reduced nuclear concentration of ligand in all cortical layers and demonstrated the specificity of [125I]MIE2 for the estrogen receptor. These results show the extensive presence of estrogen target cells in the early postnatal cortex and a profound change in topography and number of target cells during the postnatal period. These findings further suggest an important role for estrogen in the development of certain cortical neurons, possibly involving neuronal differentiation, cell positioning, and connectivity.

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.

Expression of estrogen receptor-beta messenger ribonucleic acid in oxytocin and vasopressin neurons of the rat supraoptic and paraventricular nuclei.

The regulatory actions of estrogen on magnocellular oxytocin (OT) and vasopressin (VP) neurons of the paraventricular (PVN) and supraoptic (SON) nuclei are well documented. To date it is still debated whether the effect of estrogens is exerted directly or mediated by estrogen-sensitive interneurons. Previous immunocytochemical (ICC) and in situ hybridization (ISH) studies detected either low levels or absence of the classical estrogen receptor (ER-alpha) in the PVN and the SON of the rat. The present experiments using a combined ICC and ISH method were undertaken to examine the expression of the recently cloned beta form of ER (ER-beta) in OT- and VP-immunoreactive (IR) neuronal systems of the rat hypothalamus. The results demonstrate that the highest cellular levels of ER-beta messenger RNA (mRNA) in OT-IR neurons can be visualized in the caudal portion of the PVN and in an area ventro-medial to the central core of VP-IR cells. These neurons were previously shown to project caudally to the brain stem and the spinal cord to regulate autonomic functions. In addition, the whole rostro-caudal extent of the PVN and the SON contained OT-IR neurons that coexpressed variable levels of ER-beta mRNA. Similarly, the presence of ER-beta mRNA was seen in a large population of VP-IR paraventricular and supraoptic neurons. In the SON, somewhat stronger hybridization signal was detected in VP-IR neurons as compared with OT-IR neurons. Together, these findings provide strong support for the concept that the functions of OT- and VP-IR neurons in the PVN and the SON are regulated directly by estrogen and that the genomic effects of estrogens are mediated by ER-beta.

Progestin receptor cells in the 8-day-old male and female mouse cerebral cortex: autoradiographic evidence for a sexual dimorphism in target cell number.

The present study examined the number and distribution of progestin receptor cells in the 8-day-old male and female cortex and compared cortical labeling with that in the preoptic area and central hypothalamus. Eight-day postnatal mice (four males and four females), treated with estradiol, were each sc injected with 0.32 micrograms/100 g BW [125I]progestin (SA, 2200 Ci/mM). Brains were frozen 2 h after injection of [125I]progestin, sectioned, and processed for thaw-mount autoradiography. Cells with a nuclear concentration of radioactivity were localized in lamina VI of the lateral cortical regions of the male and female brain, while only a few cortical cells were seen in laminae II, III, and V of the suprarhinal, lateral, and cingulate/paracingulate regions. Comparison of the number of labeled cells revealed that the female cortex contained significantly more labeled cells than the male at three of the four levels investigated. Similarly, the number of target cells was higher in the female medial preoptic nucleus, but not in the arcuate nucleus and ventromedial hypothalamic nucleus, while the distributions of labeled cells in the male and female preoptic/hypothalamic regions were comparable. Injection of unlabeled progesterone or R5020 1 h before [125I]progestin reduced the nuclear concentration of radioactivity in all target regions and verified the specificity of [125I]progestin for the progestin receptor. The results of these studies indicate that mouse 8-day-old cortex and preoptic area in the female animal have more progestin receptor cells than those in the male and demonstrate that progestin receptor cells are localized in a region of the cortex known to contain few estrogen target cells. These results further suggest that a sexual dimorphism in progestin cell number may result in a differential effect of progestin on the cortex and preoptic area of the mouse, perhaps establishing a dimorphism in development and function.

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.

Expression of growth differentiation factor-9 messenger ribonucleic acid in ovarian and nonovarian rodent and human tissues.

Growth differentiation factor-9 (GDF-9) is a member of the transforming growth factor-beta family that is reported to be expressed exclusively in the ovary, specifically in the oocyte. Female mice deficient in GDF-9 are infertile, suggesting that GDF-9 receptor agonists and antagonists may specifically modulate fertility. We now report that GDF-9 messenger RNA (mRNA) is expressed in nonovarian tissues in mice, rats, and humans. GDF-9 mRNA was detected in mouse and rat ovary, testis, and hypothalamus by Northern blot and RT-PCR analyses. The localization of GDF-9 mRNA specifically in oocytes of the mouse ovary was confirmed by in situ hybridization histochemistry. In mouse testis, although localization in Sertoli cell cytoplasm could not be ruled out, mRNA expression was observed in large pachytene spermatocytes and round spermatids. The expression of GDF-9 mRNA in human tissues was assessed by Northern blot and RT-PCR analyses. GDF-9 mRNA was observed in ovary and testis and, surprisingly, in diverse nongonadal tissues, including pituitary, uterus, and bone marrow. Therefore, GDF-9 mRNA expression in rodents is not exclusive to the ovary, but includes the testis and hypothalamus. Furthermore, human GDF-9 mRNA is expressed not only in the gonads, but also in several extragonadal tissues. The function and relevance of nongonadal GDF-9 mRNA are not known, but may affect strategies for contraception and fertility that are based on GDF-9 activity.

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.

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.

The ontogeny of GAP-43 (neuromodulin) mRNA in postnatal rat brain: evidence for a sex dimorphism.

GAP-43 is a membrane-bound protein selectively concentrated in axonal growth cones during brain development and implicated in axonal outgrowth and elongation. A sex dimorphism in the number of synapses in certain regions of the adult rat brain has been attributed to differences in gonadal steroid hormone action during early postnatal life. The results of recent studies have demonstrated that gonadal steroids modulate GAP-43 mRNA in regions of the postnatal and adult brain where steroid hormone receptors are concentrated. Since gonadal steroids influence the development of the sexually undifferentiated brain during the first few weeks of postnatal life, the present study investigated the ontogeny of GAP-43 mRNA in the male and female rat brain between postnatal days 1 and 25. On postnatal days 1, 3, 6, 12, 18, and 25, brains were collected from male and female postnates and frozen, and 16 microns cryostat sections were processed and hybridized with a 35S-labeled antisense riboprobe complementary to GAP-43 mRNA. Evaluation of film autoradiograms demonstrated a widespread distribution of GAP-43 mRNA in postnatal brain regions, including the cerebral cortex; bed nucleus of the stria terminalis; and medial preoptic area, ventromedial nucleus, and arcuate nucleus of the hypothalamus. Densitometric measurements revealed that GAP-43 mRNA was transiently elevated during early postnatal life, with a subsequent decrease during brain maturation, although the pattern of change varied among the brain regions investigated. In addition, the level of GAP-43 hybridization signal was significantly higher in the male cortex, bed nucleus, and medial preoptic nucleus, but not the ventromedial and arcuate nuclei, than in postnatal females. Analysis of slide autoradiograms demonstrated that the change in GAP-43 mRNA during postnatal development was due to changes at the cellular level. The present results indicate that expression of GAP-43 mRNA is transiently elevated and sexually dimorphic in certain regions of the early postnatal rat brain. The results further suggest that the differential expression of GAP-43 in the male and female postnatal brain may be related to sex differences in neuronal outgrowth and connectivity resulting in a dimorphism in the pattern of adult neuronal circuitry.

In situ hybridization analysis of the distribution of neurokinin-3 mRNA in the rat central nervous system.

The tachykinin family of neuropeptides, which includes substance P, neurokinin A, and neurokinin B, have three distinct receptors: NK-1, NK-2, and NK-3. With the cloning of the rat NK-3 cDNA, it is now possible to evaluate the distribution of NK-3 mRNA in the rat brain. Female rat brains were sectioned and hybridized with a riboprobe complimentary to NK-3 mRNA. The results of these studies revealed an extensive distribution of NK-3 mRNA throughout the rostral-caudal extent of the brain, spinal cord, and retina. In agreement with previous binding studies, we observed NK-3 mRNA in the cortex, the amygdala, the hippocampus, the medial habenula, the zona incerta, the paraventricular and supraoptic nuclei of the hypothalamus, the substantia nigra, the ventral tegmental area, the interpeduncular nucleus, the raphe nuclei, the dorsal tegmental nucleus, and the nucleus of the solitary tract. In contrast with binding data, only a few NK-3 mRNA cells were detected in the striatum. In addition, the present study detected NK-3 mRNA in the olfactory bulb, the dentate gyrus and subiculum, the medial septum, the diagonal band of Broca, the ventral pallidum, the globus pallidus, the bed nucleus of the stria terminalis, the arcuate, the premammillary and mammillary nuclei, the dorsal and lateral regions of the posterior hypothalamus, the central gray, the cerebellum, the parabrachial nuclei, the nucleus of the spinal trigeminal tract, the dorsal horn of the spinal cord, and the retina. The results of these in situ hybridization histochemical studies have provided detailed and novel information about the distribution of NK-3 mRNA and have elucidated the putative sites of neurokinin B action in the rat central nervous system.

Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system.

Estrogen plays a profound role in regulating the structure and function of many neuronal systems in the adult rat brain. The actions of estrogen were thought to be mediated by a single nuclear estrogen receptor (ER) until the recent cloning of a novel ER (ER-beta). To ascertain which ER is involved in the regulation of different brain regions, the present study compared the distribution of the classical (ER-alpha) and novel (ER-beta) forms of ER mRNA-expressing neurons in the central nervous system (CNS) of the rat with in situ hybridization histochemistry. Female rat brain, spinal cord, and eyes were frozen, and cryostat sections were collected on slides, hybridized with [35S]-labeled antisense riboprobes complimentary to ER-alpha or ER-beta mRNA, stringently washed, and opposed to emulsion. The results of these studies revealed the presence of ER-alpha and ER-beta mRNA throughout the rostral-caudal extent of the brain and spinal cord. Neurons of the olfactory bulb, supraoptic, paraventricular, suprachiasmatic, and tuberal hypothalamic nuclei, zona incerta, ventral tegmental area, cerebellum (Purkinje cells), laminae III-V, VIII, and IX of the spinal cord, and pineal gland contained exclusively ER-beta mRNA. In contrast, only ER-alpha hybridization signal was seen in the ventromedial hypothalamic nucleus and subfornical organ. Perikarya in other brain regions, including the bed nucleus of the stria terminalis, medial and cortical amygdaloid nuclei, preoptic area, lateral habenula, periaqueductal gray, parabrachial nucleus, locus ceruleus, nucleus of the solitary tract, spinal trigeminal nucleus and superficial laminae of the spinal cord, contained both forms of ER mRNA. Although the cerebral cortex and hippocampus contained both ER mRNAs, the hybridization signal for ER-alpha mRNA was very weak compared with ER-beta mRNA. The results of these in situ hybridization studies provide detailed information about the distribution of ER-alpha and ER-beta mRNAs in the rat CNS. In addition, this comparative study provides evidence that the region-specific expression of ER-alpha, ER-beta, or both may be important in determining the physiological responses of neuronal populations to estrogen action.