| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Atherosclerosis and Endocrinology, Merck Research Laboratories (S.W.M., J.Y., L.P., H.A.W., S.P.R., J.M.S., S.E.A.), Rahway, New Jersey 07065; Laboratory of Endocrinology, Graduate School of Integrated Science, Yokohama City University (S.H.), Yokahama 236-0027, Japan; and Departments of Neuroendocrinology (E.H., B.S.M.) and Neurobiology and Behavior (D.W.P., S.O.), Rockefeller University, New York, New York 10021
Address all correspondence and requests for reprints to: Stephen E. Alves, Ph.D., Department of Atherosclerosis and Endocrinology, Merck Research Laboratories, 126 Lincoln Avenue (RY 80T-126), Rahway, New Jersey 07065. E-mail: stephen_alves{at}merck.com.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
(ER) and ERß are members of the steroid nuclear receptor family that modulate gene transcription in an estrogen-dependent manner. ER mRNA and protein have been detected both peripherally and in the central nervous system, with most data having come from the rat. Here we report the development of an ERß-selective antibody that cross-reacts with mouse, rat, and human ERß protein and its use to determine the distribution of ERß in the murine brain. Further, a previously characterized polyclonal antibody to ER was used to compare the distribution of the two receptors in the first comprehensive description of ER distribution specifically in the mouse brain. ERß immunoreactivity (ir) was primarily localized to cell nuclei within select regions of the brain, including the olfactory bulb, cerebral cortex, septum, preoptic area, bed nucleus of the stria terminalis, amygdala, paraventricular hypothalamic nucleus, thalamus, ventral tegmental area, substantia nigra, dorsal raphe, locus coeruleus, and cerebellum. Extranuclear-ir was detected in several areas, including fibers of the olfactory bulb, CA3 stratum lucidum, and CA1 stratum radiatum of the hippocampus and cerebellum. Although both receptors were generally expressed in a similar distribution through the brain, nuclear ER-ir was the predominant subtype in the hippocampus, preoptic area, and most of the hypothalamus, whereas it was sparse or absent from the cerebral cortex and cerebellum. Collectively, these findings demonstrate the region-selective expression of ERß and ER in the adult ovariectomized mouse brain. These data provide an anatomical framework for understanding the mechanisms by which estrogen regulates specific neural systems in the mouse. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
(ER) and ERß, members of the steroid receptor branch of the nuclear receptor superfamily. ERs, when bound to agonists such as 17ß-estradiol, dimerize and interact with regulatory DNA sequences to modulate gene transcription by interacting with coactivators and the basal transcriptional machinery (2, 3). Further, increasing evidence indicates rapid, nongenomic actions of estrogen, and some of these effects may be mediated through extranuclear ER and/or ERß (4, 5, 6, 7, 8).
The CNS is a major target of estrogen action (9, 10), yet the mechanisms by which estrogen mediates these effects are not clear. Knowledge of the distribution of ERs across specific brain regions provides insight into estrogen target sites and the complex regulatory actions this class of steroid hormones has on brain function. To date, the most extensive information regarding ER expression patterns in the CNS has come specifically from the rat (11, 12, 13, 14, 15, 16, 17, 18). A comprehensive study comparing ER and ERß mRNA expression patterns in rat CNS has indicated some overlap, but also some striking differences in the distribution and abundance of expression of these two receptors (14). Molecular studies suggest that the two ER isoforms can play different roles in estrogen regulation of gene expression and neuronal physiology (2, 19, 20, 21, 22). Further, a comparison of ER mRNA expression patterns in rat (11, 14, 23), mouse (24, 25), monkey (26), and human (27, 28) indicates that data cannot be extrapolated from one species to another.
The mouse has become an increasingly valuable species to identify specific molecular mediators of steroid hormone actions, largely due to the availability of transgenic/knockout models (29, 30). Shughrue and colleagues (24, 31) were the first to report ERß mRNA and [125I]estradiol binding specifically in the forebrain of the ER knockout (ERKO) mouse as evidence for the expression of functional ERß protein in select forebrain regions. However, this group has more recently identified low levels of estrogen binding and regulation of gene expression in the preoptic area of the double ßERKO mouse (32), activity attributed to an ER splice variant specifically produced in the ERKO and previously identified in uterus (33). Collectively, these findings underscore the importance of using subtype-selective antibodies to confirm the distribution of ER and ERß protein in the mouse brain.
The ER gene was cloned 10 yr before the identification of ERß (34, 35); thus, many more antibodies to the ER have been generated (36). In the present study we describe the characterization of a polyclonal peptide antibody (80424) raised against a conserved region of the human, mouse, and rat ERß protein recently used to identify a longer isoform of human ERß-548 (37). Using the 80424 antibody, we describe the distribution of ERß immunoreactivity (-ir) in the mouse brain. Further, a previously characterized antibody to ER (AS409; Refs. 38, 39, 40, 41) was used to compare the distributions of the two receptor subtypes in the ovariectomized mouse brain.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
Immunoblotting
Extracts of SF9 cells expressing rat ER (595 aa), human ER (595 aa), mouse ERß (485 aa), rat ERß (485 aa), human ERß (485 aa), and ERß (530 aa) were obtained from Stefan Nilsson (Karo Bio, Huddinge, Sweden). Human tissues were obtained from the National Disease Research Interchange (Philadelphia, PA). Extracts from tissues and cells were reduced and denatured in SDS/mercaptoethanol by heating to 75 C for 20 min, then subjected to SDS-PAGE using ready made gels [Bio-Rad Laboratories, Inc. (Hercules, CA) and Novex (San Diego, CA)]. Separated proteins were transferred to polyvinylidene difluoride membranes by electrophoresis and blocked with blocking buffer [10 mM phosphate (pH 7.5), 0.1 M NaCl, 10% glycerol, 10% nonfat milk, and 0.2% Tween 20]. Blots were incubated at room temperature with the antibody at a 1:2000 dilution for 16 h. Unbound antibody was removed by repeated washing with blocking buffer, and cells were treated with horseradish peroxidase-conjugated antirabbit antibody (Sigma-Aldrich, Milwaukee, WI) at a 1:2000 dilution for 1 h at room temperature. The blots were developed with Supersignal West Dura ECL substrates (Pierce, Rockford, IL) according to the manufacturer’s instructions. Total extracts from mouse and rat brain and human ovary and testes were immunoblotted with a 1:200 dilution of antibody. Peptide competition was performed by incubating 200 ng peptide immunogen/µl antibody for 1 h at room temperature before use.
Immunofluorescence microscopy
COS-7 cells were transiently transfected with ER and ERß to initially determine the sensitivity and selectivity of 80424 in immunocytochemistry. Transfected COS-7 cells were fixed with 3% paraformaldehyde in PBS. Cells were permeabilized with 1% Triton X-100 in PBS, and nonspecific binding was blocked with normal goat serum. Cells were incubated in the anti-ERß primary antibody, 80424, at a 1:1000 dilution. Fluorescein isothiocyanate-conjugated antirabbit antibody was used as the secondary antibody at a dilution of 1:2000. Slides were examined under a Nikon light microscope (Melville, NY), and images were captured with a Hamamatsu digital camera (Hamamatsu City, Japan) and Metamorph software (Universal Imaging Corp., Downingtown, PA).
Animal treatment and tissue preparation
Animal care, maintenance, and surgery were performed in accordance with regulations dictated by the Animal Welfare Act and the U.S. DHHS Guide for the Care and Use of Laboratory Animals. Young adult female mice derived from crosses of C57BL/6J and 129 ancestry were ovariectomized under Metofane anesthesia. Two weeks later, animals were deeply anesthetized with Metofane and were transcardially perfused with 100 ml 3.75% acrolein and 2% paraformaldehyde in 0.1 M sodium phosphate buffer (PB), pH 7.4. Brains were immediately removed and postfixed in 2% paraformaldehyde in 0.1 M PB at 4 C overnight. Brains were transferred into 30% sucrose in 0.1 M PB, frozen on dry ice, and sectioned at 30 µm on a sliding microtome. Sections were stored in cryoprotectant (30% glycerol and 30% ethylene glycol in 0.1 M PB, pH 7.4) at -20 C until processing for immunocytochemistry.
Immunocytochemistry
Free-floating, 30-µm sections were washed in cold 0.1 M PB with saline (PBS), pH 7.4, to thoroughly remove cryoprotectant. To ensure consistent immunolabeling across animals, a screen-bottom, 24-well tissue holder and fitted solid Lucite tray containing buffer were used. Thus, all sections were coincubated in the same solutions/conditions during the entire experiment. To deter nonspecific staining in acrolein-fixed tissue (remove excess aldehyde groups), sections were washed in 1% sodium borohydride (NaBH4) in PBS for 30 min and rinsed 8–10 times with PBS. Endogenous peroxidase activity was inhibited by washing sections in 0.3% hydrogen peroxide and 20% methanol in PBS for 15 min. Sections were washed several times in PBS and then blocked with 2% normal serum in PBS with 0.2% Triton X-100 for 1 h. Sections were incubated with the rabbit polyclonal ERß antibody (80424; 1:14,000; stock, 4.8 mg/ml) over 3 nights in blocking buffer diluted to 1% normal serum and 0.1% Triton X-100 at 4 C. Several controls were included to ensure the specificity of the immunocytochemical labeling with the 80424 antibody. A subset of sections was incubated 1) in primary antisera that had been preabsorbed with the antigenic synthesizing peptide (1:1, wt/vol); 2) in antibody preabsorbed with a nonantigenic peptide sequence of the ERß (1:1, wt/vol), or 3) without primary antibodies. Adjacent sections were incubated with an extensively characterized rabbit polyclonal antibody to ER (AS409; 1:20,000) (38, 39, 40, 41) over 5 nights to compare the distribution of ER. We recently demonstrated the specificity of this antibody for the ER in mouse brain; no immunolabel was observed in the ERKO dorsal raphe (40), a region with abundant nuclear ERß. Sections were washed in PBS and exposed to a biotinylated secondary goat antirabbit antibody (1:600; Vector Laboratories, Inc., Burlingame, CA) in PBS with 0.2% Triton X-100 and 1% normal serum for 1 h. After several PBS washes, tissue was exposed to the avidin-biotin complex (ABC Elite kit, Vector Laboratories, Inc.) in PBS for 30 min. Sections were rinsed in PBS and incubated in tyramide signal amplification (TSA) solution (1:50; NEN Life Science Products, Boston, MA) for 10 min, washed in PBS, and then incubated with streptavidin-horse radish peroxidase (1:200) for 30 min. A subset of tissue was not incubated with the TSA system for comparison. Sections were washed in PBS, followed by 0.175 M sodium acetate buffer (pH 7.0; Sigma-Aldrich, St. Louis, MO), and then exposed to the substrate 3,3'-diaminobenzidine tetrachloride containing nickel sulfate and hydrogen peroxide in 0.175 M sodium acetate buffer for 3 min. The reaction product appears as a dark blue-black punctate stain, primarily in cell nuclei. After one sodium acetate buffer rinse and several PBS washes, sections were mounted onto gelatin-coated slides in 0.05 M PB, air-dried overnight, dehydrated in ascending ethanol concentrations, cleared in xylene, and coverslipped with DPX mounting medium. Photographs were taken under a Nikon light microscope and 35-mm camera (Nikon, Tokyo, Japan) using Kodak Ektachrome slide film (Eastman Kodak Co., Rochester, NY). Slides were scanned and saved to a Macintosh computer (Cupertino, CA), imported into Adobe Photoshop (Adobe Systems, Mountain View, CA) for labeling.
Tissue analysis
Sequential 30-µm sections, approximately 180 µm apart, from the olfactory bulbs through the hindbrain were immunostained as described above. Each section was anatomically categorized according to distance from bregma (B) using The Mouse Brain in Stereotaxic Coordinates (42) as a guide. The distribution and relative density of cells immunoreactive to the 80424 antibody (ERß) or AS409 (ER) were documented at magnifications of x100 and x200 using a Nikon light microscope. Data were expressed in tabular form: -, no ER-ir cells; -/+, few ER-ir cells; +, ER-ir cells present; 2+, abundant ER-ir; and 3+, very abundant ER-ir. Maps were generated to depict the distribution of nuclear 80424 (ERß)-ir, as ERß localization with this affinity-purified antibody was the primary focus of this study.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
). This sequence is located within the A/B domain of ERß (exons 2–3) and is not conserved in ER. Accordingly, this peptide was selected to generate rabbit polyclonal antibodies that would be subtype selective, but not species specific. The serum from one rabbit (designated 80424) was peptide affinity-purified and used for the analyses described below.
|
, lanes 1 and 2) and human ERß (Fig. 1C, lanes 1 and 2) identified a band migrating at approximately 60 kDa, whereas no staining was detectable in extracts of cells transfected with either rat or human ER (Fig. 1B, lanes 3 and 4). A band migrating at approximately 55 kDa was detected in extracts of human ovary and human testes (Fig. 1C, lanes 3 and 4). The latter showed an additional band migrating with a mobility of approximately 70 kDa. Upon immunoblotting whole tissue extracts from the rat and mouse brain, a single major band migrating with the mobility of about 70 kDa was detected (Fig. 1C, lanes 5 and 6). No bands were detected in tissue extracts or in extracts of SF9 cells expressing cloned mouse, rat, or human ERß when 80424 was preincubated with the immunogenic peptide (Fig. 1B, lanes 5 and 6, and Fig. 1C, lanes 7–12). Therefore, 80424 selectively recognizes this peptide sequence in mouse, rat, and human ERß and proteins of similar size in whole tissue extracts.
A population of the COS-7 cells transiently transfected with human ERß showed strong nuclear staining by immunofluorescence with the 80424 antibody, and this labeling could be eliminated by prior incubation with the immunogenic peptide (Fig. 2). No staining was detectable in ER-transfected cells or in cells transfected with an empty vector (data not shown). Therefore, 80424 was determined to be suitable for immunocytochemical studies.
|
. The majority of immunostaining was detected specifically within cell nuclei, as demonstrated in the locus coeruleus (Fig. 3, A and C); however, cells within select regions consistently exhibited extranuclear (somal and/or fiber) labeling (Fig. 3, D and F). Importantly, no staining was present when tissue was incubated with antibody that had been preadsorbed with the antigenic peptide (Fig. 3, B and E). When the 80424 antibody was preincubated with a peptide from an alternate region of ERß, immunolabeling was indistinguishable from that seen in tissue incubated with antibody alone (Fig. 3C). The TSA system enhanced signal detection.
|
. Table 1 is a comprehensive list of the specific brain regions observed to exhibit ERß-ir and/or ER-ir and the subcellular distribution of immunolabel.
|
|
); striatum; ventral pallidum; subiculum; CA3 region of the hippocampus; internal capsule; substantia innominata; nucleus accumbens; cortical, amygdaloid, and central nuclei of the amygdala; several thalamic nuclei (central medial, rhomboid, and habenular); and the subincertal nucleus. Similar levels of nuclear ERß-ir were observed in cells of the anterior and lateral hypothalamic areas; the arcuate, periventricular, supraoptic, and suprachiasmatic nuclei; the tuber cinereum area; and the ventrolateral division of the ventromedial nucleus. Interestingly, cells within the most caudal extent of the arcuate and ventromedial nuclei exhibited darker nuclear ERß-ir compared with the rest of these nuclei, in which low to moderate labeling was seen (Fig. 4; levels, -1.34 and -1.70).
In most brain areas ERß-ir was observed distinctly within the nuclear compartment of positive cells. Such nuclear-ir ranged from extremely dense, punctate stain, as seen in the medial amygdala and dorsal raphe (Fig. 5, B and D), to weak nuclear-ir, as observed in scattered cells within the CA3 pyramidal layer of the hippocampus (Fig. 3F). In the CA3 stratum lucidum, cytoplasmic labeling was observed in cross-sectional fibers (Fig. 3F). In the olfactory bulb, while nuclear-ir was seen within small cells of the glomerular layer, distinct cytoplasmic fiber labeling were observed in the external plexiform layer (Fig. 5A). Less intense fiber labeling was seen through the CA1 stratum radiatum of the hippocampus (data not shown).
|
(locus coeruleus) and Fig. 5 (substantia nigra and dorsal raphe nucleus). Areas observed to have moderate levels of ERß-ir included the dorsal terminal and Edinger-Westphal nuclei; the compact and lateral divisions of the substantia nigra (caudal); the parabrachial pigmented nucleus; the rostral linear raphe; B9 serotonin cells; pontine and pontine reticular nuclei; subbrachial and paralemniscal nuclei; the lateral lemniscus; the parabigeminal, laterodorsal tegmental, reticulotegmental, and anterior tegmental nuclei; the medioventral periolivary nucleus; and the periolivary nuclei. Some nuclear ERß-ir also was seen in the deep mesencephalic, interfascicular, and the lateral and medial vestibular nuclei. Interestingly, although distinct fiber labeling was seen in the dorsal medial division of the periaqueductal gray above the central aqueduct, little or no somal or nuclear ERß-ir was observed in this brain region.
Hindbrain/cerebellum.
Cells exhibiting strong nuclear ERß-ir were recorded in the medullary raphe nuclei (magnus, pallidus, and obscurus) and the dorsal motor 10 nucleus of vagus. Moderate ERß-ir levels were observed in the parapyramidal, gigantocellular reticular (), rostroventrolateral reticular inferior olive (principle nucleus), lateral reticular nucleus, and spinal 5 nucleus (oral). In the cerebellum, weak to moderate nuclear ERß-ir was observed in the pyramidal cell layer and also scattered within granule cells (Fig. 5F). Fiber/somal label was also evident in the cerebellum. In the remaining hindbrain, scattered cells exhibiting immunolabel were seen in the supratrigeminal nucleus, intermediate reticular nucleus, prepositus nucleus, and the nucleus ambiguous. Somal labeling was observed within cells of the facial nucleus.
Dark nuclear label also was identified within cells interspersed within areas of white matter, including the facial nerve and the cerebellar peduncle. These cells could be infiltrated neurons scattered within the myelinated tracts and/or glial cells such as oligodendrocytes.
ER localization in brain using AS409 antibody
The general distribution patterns of nuclear ER-ir and ERß-ir in the mouse brain were similar, although there were differences in the abundance of receptor labeling in many overlapping regions as well as several areas that exhibited distinct expression of one receptor isoform. The selectivity of AS409 and 80424 antibodies to distinguish ER or ERß, respectively, in mouse brain, is demonstrated in Fig. 6. The AS409 antibody specifically recognized cells in the lateral septum that appeared to exclusively express ER, whereas 80424 detected cells that express only ERß within the medial septum and vertical limb of the diagonal band.
|
-ir included the BNST, medial amygdala, medial preoptic nucleus, arcuate nucleus, and ventrolateral division of the ventromedial nucleus of the hypothalamus. Other areas with pronounced nuclear ER-ir included the islands of Calleja, lateral septum, septohippocampal nucleus, ventral hippocampus, amygdalohippocampal nucleus, anteroventral periventricular nucleus, medial preoptic area, dorsomedial and periventricular nuclei of the hypothalamus, and the central division of the ventromedial nucleus. Scattered cells exhibiting nuclear ER-ir were recorded in the granule cell layer of the olfactory bulb; several cortical areas (granular insular, infralimbic, perirhinal, and second somatosensory cortexes); striatum (caudate putamen); ventral pallidum; subfornical organ; dorsal hippocampus; internal capsule; substantia innominata; anterior, basal, and central amygdaloid nuclei; and the precommisural, pretectal, and lateral posterior thalamic nuclei. In the preoptic area-hypothalamus, scattered nuclear ER-ir was seen in the lateral and magnocellular preoptic areas, anterior commissural nucleus, anterior hypothalamic area, lateral hypothalamic area, medial tuberal nucleus, PVN, posterior hypothalamic area, supraoptic nucleus, tuber cinereum area, dorsomedial ventromedial nucleus, and across the mammillary nuclei.
Midbrain/pons.
The periaqueductal gray and the lateral parabrachial nucleus exhibited the most abundant nuclear ER-ir at this brain level. Other regions of the midbrain/pons that exhibited moderate to low levels of ER-ir included the deep mesencephalic, dorsal terminal, and interfascicular nuclei; compact subdivision of the substantia nigra; inferior colliculus; midbrain raphe nuclei; pontine reticular, intercollicular, subbrachial, and laterodorsal tegmental nuclei; trapezoid body; locus coeruleus; medial parabrachial nucleus; and dorsal periolivary region. Some ER-ir also was recorded in the interpeduncular nucleus; lateral division of the substantia nigra; superior colliculus; nucleus brachium inferior colliculus; B9 serotonin cells; pedunculopontine tegmental, medioventral periolivary, subcoeruleus, and intermediate reticular nuclei; periolivary nuclei; and prepositus nucleus.
Hindbrain/cerebellum.
The nucleus of the solitary tract exhibited the most robust nuclear ER-ir in the hindbrain. Other areas with distinct ER-ir included the posterodorsal tegmental nucleus; supratrigemenal, parapyramidal, gigantocellular and paragigantocellular reticular nuclei; area postrema; rostroventrolateral reticular and lateral reticular nuclei; and dorsomedial spinal 5 and spinal 5 nuclei. Further, some nuclear ER-ir was seen in the intermediate reticular and prepositus nuclei, dorsal motor 10 nucleus of vagus, and the spinal 5 nucleus, oral division. No distinct ER-ir was observed in the cerebellum.
As was seen for ERß-ir, the majority of ER-ir was distinctly within cell nuclei. Across various regions of the brain, some somal immunostaining could be seen as a halo around positively labeled nuclei (data not shown). In general, there was far less distinct extranuclear ER-ir, such as fiber labeling, compared with what was seen for ERß-ir in select brain regions. However, ultrastructural evidence indicates that this receptor can be found in discrete extranuclear sites (dendritic spines, axons, and axon terminals) in the rodent brain (5, 8).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
antibody, to document the localization of these ER subtypes in the brain of the ovariectomized mouse. This work represents the most comprehensive description of ER distribution specifically in the murine CNS. Although the distribution patterns of both receptor subtypes are generally similar, several brain regions exhibit a preferential expression of one isoform, and some regions appear to express only one ER subtype. Data from select brain regions will be highlighted below, and our findings will be compared with previously reported data in the rat and mouse.
Specificity of the 80424 antibody
The novel polyclonal ERß peptide antibody 80424 selectively recognizes ERß in mouse, rat, and human tissue, as determined by immunoblot analysis and immunocytochemistry in transfected cells and in mouse brain sections. The apparent size of the approximately 70-kDa band detected in rodent brain and human testicular extracts appears to be larger than the expected size of cloned ERß and is suggestive of possible posttranslational modifications, including acetylation, phosphorylation, and/or glycosylation. Further, the major band seen in extracts of the human ovary (
55 kDa) and the second band seen in the testicular extracts were slightly smaller than the predicted size (60 kDa) of recombinant human ERß, perhaps as a result of posttranslational modifications. Immunocytochemical staining in transfected COS-7 cells was localized specifically to cell nuclei.
A number of ERß isoforms have been reported in most or all species examined (43, 44, 45, 46, 47). In the mouse several isoforms have been identified with altered ligand-binding domains, deletions of exon 5 or 6 or insertion of an additional 54 nucleotides between exons 5 and 6 (46, 47), thus significantly reducing estrogen binding. The 80424 antibody does not distinguish between these alternate isoforms, as the epitope spans exons 2 and 3. Thus, the distribution of ERß-ir that we are reporting in the mouse brain presumably represents both high and low affinity (or nonbinding) receptors. Therefore, this distribution may not completely overlap with estrogen binding data in the mouse brain.
Comparison of ER subtype distribution in the mouse brain
A comparison of ER-ir and ERß-ir in the brain of the adult ovariectomized mouse provides insight into the role each ER subtype may play in murine brain function. However, as ER expression patterns are likely to vary in the developing, intact cycling adult and/or aging female brain, these data should be considered to represent ER distribution specifically in the estrogen-depleted young adult murine brain. Although the expression of ER is estrogen regulated (39), less is known regarding estrogen modulation of ERß expression in the CNS. For example, although estrogen replacement did not diminish ERß mRNA in brain regions examined in the spayed macaque (26), some rodent studies have reported estrogen regulation of ERß mRNA or protein in a region-specific manner (23, 48, 49, 50).
In the forebrain, areas such as the BNST and the caudal extent of the medial amygdala exhibited nearly equally abundant nuclear-ir to ER and ERß. Although robust ER-ir was found throughout the extent of the medial amygdala, ERß-ir cells were most heavily concentrated caudally. The abundance of both ERs in the caudal aspect of the medial amygdala suggests that the two receptors may be expressed in the same cells and perhaps form functional heterodimers, as has been demonstrated in vitro (51, 52). Both ER subtypes have been reported to colocalize in the rat medial amygdala (48, 53) as well as the anteroventral periventricular nucleus of the preoptic area (18, 48).
Throughout the cerebral cortex, ERß was observed to be the predominant receptor subtype; most cells exhibited light to moderate nuclear-ir. However, there were scattered cells with darkly labeled nuclei, particularly those found adjacent to the external capsule and cingulum and scattered in the piriform cortex. The ERß also has been reported to be the predominant ER subtype in the cerebral cortex of the rat (14); however, the expression pattern differs greatly between these two rodent species. In the rat there is a more extensive and consistent expression pattern of strong nuclear label throughout laminae IV and VI (13), whereas in the mouse cortex, immunolabeling is found in select subregions (i.e. somatosensory and insular regions) and is predominantly low to moderate in intensity.
The basal forebrain exhibited both ER subtypes in varying levels. In the striatum, scattered cells exhibited light to moderate nuclear-ir for both receptors, as previously reported in a developmental study of the mouse striatum (25), but the ER-ir was generally more intense. Moderate to intense nuclear ERß-ir was observed in cells within the globus pallidus, whereas little ER-ir was found in this brain region. Within the septum, ER was found specifically within cells of the lateral nucleus, whereas ERß-ir was observed almost exclusively within cells in the medial nucleus, a largely cholinergic region. Both the horizontal and vertical limbs of the diagonal band of Broca, other major cholinergic areas, contained many cells with intense ERß-ir, whereas little ER-ir was observed in these regions. Once again the mouse appears to differ from the rat, as these basal forebrain regions in the rat express both ER subtypes in abundance (14), and nearly all of the ER-expressing cholinergic neurons in the rat have been reported to exhibit specifically ER-ir (54).
Both receptor subtypes were observed in the preoptic area and hypothalamus, with regional variations. ER predominated in the medial preoptic area and the arcuate and ventromedial nuclei of the hypothalamus, regions important in the regulation of reproductive physiology and behavior. These findings are consistent with the profound reproductive abnormalities seen specifically in the ERKO mouse (2, 20, 55, 56, 57). The ERß was more abundant in the magnocellular preoptic area and the PVN. The rat PVN was first identified as a major site of ERß expression (14, 15, 16, 17). Although the rat PVN has been reported to express only ERß (14), we have found that the murine PVN contains cells that express moderate levels of ER. Thus, both ER subtypes may contribute to estrogen’s actions in the mouse PVN. Interestingly, the human PVN also has been reported to express mRNA for both ER and ERß; in fact, ER appears to be the predominant subtype in the human PVN (27).
Within the midbrain, pons, and brainstem, a predominance of ERß was observed in several of the major monoaminergic cell populations. These include the heavily dopaminergic substantia nigra and ventral tegmental area, the serotonergic raphe nuclei, and the noradrenergic locus coeruleus. Although light to moderate ER-ir also was observed in some of these regions, the number and intensity of cells exhibiting nuclear ERß-ir were far more abundant. These findings suggest that estrogen is likely to regulate these monoaminergic systems primarily through the ERß. Regarding the dorsal raphe nucleus, this appears to be consistent across species examined, specifically the mouse (40), rat (14, 58), macaque (26), and guinea pig (59). The cerebellum of the adult mouse appears to be similar to that of the adult rat (14), as only ERß-ir was seen in this brain region, although the intensity of immunolabel appeared to be generally weaker in the mouse compared with what has been reported in the rat (15).
Our data for ERß-ir in mouse brain are generally in agreement with a previous report describing the distribution of ERß mRNA in the forebrain and rostral midbrain of the ERKO mouse (24). Notable exceptions include the thalamus, substantia nigra, and ventral tegmental area. We observed cells with moderate to robust nuclear ERß-ir within several thalamic nuclei and robust nuclear ERß-ir in the substantia nigra and ventral tegmental area, yet no hybridization signal for ERß was reported in these regions in the ERKO mouse (24). Further, although we observed only weak ERß-ir in the suprachiasmatic nucleus, this region was reported to express abundant mRNA. Considering that we examined wild-type mice in the present study, it is conceivable that disruption of the ER gene could result in differences in ERß expression in select regions. Indeed, very recent evidence indicates that ERß expression is greatly modified in specific brain regions in the male ERKO mouse compared with wild-type siblings (50). Alternatively, these areas may express levels of mRNA that are not proportional to the transcribed protein.
Regarding extranuclear immunostaining, several areas of the mouse brain exhibited distinct fiber labeling for ERß, most notably in fibers of the olfactory bulb and in cross-sectional fibers of the CA3 stratum lucidum of the hippocampus. This labeling was absent upon preabsorption with the cognate peptide, suggesting that the signal was indeed specific for this ERß sequence. Cytoplasmic distribution of ERß-ir has been reported by several groups using other antibodies (16, 17, 60, 61, 62). In the present light microscopy study, extranuclear ER-ir was less evident, mostly appearing as light somal immunostaining surrounding intense nuclear-ir. Despite little extranuclear ER-ir evident at the light microscopic level, several studies examining the ultrastructural distribution of ER using multiple antibodies have identified this receptor in discrete subcellular compartments in the rat brain. Specifically, such labeling has been reported in axons and axon terminals in the rat hypothalamus (63, 64) and more recently in axons, axon terminals, and dendritic spines in neurons of the hippocampus (5, 8). Further studies are currently underway to determine the subcellular distribution of ERß-ir.
|
Conclusion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
in the mouse brain. This work represents the most comprehensive description of ER distribution in the murine CNS. The mouse has become an important animal model in neuroscience research based largely on the well characterized genome and the technology to generate knockout/transgenic lines in this rodent species. Integration of the present findings of ER subtype distribution into the established network of neuronal pathways provides an anatomical basis from which we may further our understanding of estrogen’s mechanisms of action on murine brain function. Further, together with data reported in other species, these findings confirm that there are both similarities and differences in expression patterns of ER and ERß in the CNS between species.
|
|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: aa, Amino acid; BNST, bed nucleus of the stria terminalis; CNS, central nervous system; ER, estrogen receptor; ERKO, ER knockout; -ir, immunoreactivity; PB, sodium phosphate buffer; PVN, paraventricular nucleus; TSA, tyramide signal amplification.
Received October 15, 2002.
Accepted for publication January 10, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
estrogen receptors are located at extranuclear sites. J Comp Neurol 429:355–371[CrossRef][Medline]
. Steroids 67:429–437[CrossRef][Medline]
in the hippocampus of female rats. J Neurosci 22:3608–3614 and -ß mRNA in the rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
and ß immunoreactivity by oxytocin neurons of rat paraventricular nucleus. J Neuroendocrinol 9:803–806[CrossRef][Medline]
and ERß at AP1 sites. Science 277:1508–1510 and -ß mRNA within the female rat brain. Brain Res Mol Brain Res 54:175–180[Medline]
knockout mouse. Endocrinology 138:5649–5652 and ß mRNA in the developing and adult mouse striatum. Neurosci Lett 276:95–98[CrossRef][Medline]
mRNA. J Clin Endocrinol Metab 85:3840–3846-knockout mice. Endocrinology 140:2613–2620 and ß. Steroids 66:1–16[CrossRef][Medline]
(ER) gene-disrupted mice. J Comp Neurol 427:185–195[CrossRef][Medline]
and progestin receptor proteins in the female rat forebrain: effects of estradiol treatment. Endocrinology 142:5172–5181 (ER) gene expression and on its ligand in a brain region-specific manner. Mol Brain Res 110:7–14[Medline]
. Mol Endocrinol 11:1486–1496 transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140:5566–5578 immunoreactivity in neurons of the rat forebrain. Endocrinology 139:5267–5270 and ERß) in the cholinergic neurons of the rat basal forebrain. Neuroscience 96:41–49[CrossRef][Medline]
gene expression in reproduction-related behaviors in female mice. Endocrinology 139:5070–5081This article has been cited by other articles:
 |
|
 |
|
  J.-R. Chen, Y.-T. Yan, T.-J. Wang, L.-J. Chen, Y.-J. Wang, and G.-F. Tseng Gonadal Hormones Modulate the Dendritic Spine Densities of Primary Cortical Pyramidal Neurons in Adult Female Rat Cereb Cortex, November 1, 2009; 19(11): 2719 - 2727. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Xue, M. Singh, F. Guo, M. Hay, and A. K. Johnson Protective actions of estrogen on angiotensin II-induced hypertension: role of central nitric oxide Am J Physiol Heart Circ Physiol, November 1, 2009; 297(5): H1638 - H1646. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. A. Penatti, D. M. Porter, and L. P. Henderson Chronic Exposure to Anabolic Androgenic Steroids Alters Neuronal Function in the Mammalian Forebrain via Androgen Receptor- and Estrogen Receptor-Mediated Mechanisms J. Neurosci., October 7, 2009; 29(40): 12484 - 12496. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. G J Hazell, S. T Yao, J. A Roper, E. R Prossnitz, A.-M. O'Carroll, and S. J Lolait Localisation of GPR30, a novel G protein-coupled oestrogen receptor, suggests multiple functions in rodent brain and peripheral tissues J. Endocrinol., August 1, 2009; 202(2): 223 - 236. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. A. Ledoux, T. Smejkalova, R. M. May, B. M. Cooke, and C. S. Woolley Estradiol Facilitates the Release of Neuropeptide Y to Suppress Hippocampus-Dependent Seizures J. Neurosci., February 4, 2009; 29(5): 1457 - 1468. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. A. Molenda-Figueira, S. D. Murphy, K. L. Shea, N. K. Siegal, Y. Zhao, J. G. Chadwick Jr., L. A. Denner, and M. J. Tetel Steroid Receptor Coactivator-1 from Brain Physically Interacts Differentially with Steroid Receptor Subtypes Endocrinology, October 1, 2008; 149(10): 5272 - 5279. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. J. Bode, M. Stephan, H. Suhling, R. Pabst, R. H. Straub, K. A. Raber, M. Bonin, H. P. Nguyen, O. Riess, A. Bauer, et al. Sex differences in a transgenic rat model of Huntington's disease: decreased 17{beta}-estradiol levels correlate with reduced numbers of DARPP32+ neurons in males Hum. Mol. Genet., September 1, 2008; 17(17): 2595 - 2609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Isken, A. F. H. Pfeiffer, R. Nogueiras, M. A. Osterhoff, M. Ristow, B. Thorens, M. H. Tschop, and M. O. Weickert Deficiency of glucose-dependent insulinotropic polypeptide receptor prevents ovariectomy-induced obesity in mice Am J Physiol Endocrinol Metab, August 1, 2008; 295(2): E350 - E355. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Milner, L. S. Lubbers, S. E. Alves, and B. S. McEwen Nuclear and Extranuclear Estrogen Binding Sites in the Rat Forebrain and Autonomic Medullary Areas Endocrinology, July 1, 2008; 149(7): 3306 - 3312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Carroll and C. J. Pike Selective Estrogen Receptor Modulators Differentially Regulate Alzheimer-Like Changes in Female 3xTg-AD Mice Endocrinology, May 1, 2008; 149(5): 2607 - 2611. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Moldovanova, C. Schroeder, G. Jacob, C. Hiemke, A. Diedrich, F. C. Luft, and J. Jordan Hormonal Influences on Cardiovascular Norepinephrine Transporter Responses in Healthy Women Hypertension, April 1, 2008; 51(4): 1203 - 1209. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zhou, A. W. Lee, N. Devidze, Q. Zhang, L.-M. Kow, and D. W. Pfaff Histamine-Induced Excitatory Responses in Mouse Ventromedial Hypothalamic Neurons: Ionic Mechanisms and Estrogenic Regulation J Neurophysiol, December 1, 2007; 98(6): 3143 - 3152. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Campbell and A. E. Herbison Definition of Brainstem Afferents to Gonadotropin-Releasing Hormone Neurons in the Mouse Using Conditional Viral Tract Tracing Endocrinology, December 1, 2007; 148(12): 5884 - 5890. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. N. Karatsoreos, A. Wang, J. Sasanian, and R. Silver A Role for Androgens in Regulating Circadian Behavior and the Suprachiasmatic Nucleus Endocrinology, November 1, 2007; 148(11): 5487 - 5495. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Tiwari-Woodruff, L. B. J. Morales, R. Lee, and R. R. Voskuhl Differential neuroprotective and antiinflammatory effects of estrogen receptor (ER){alpha} and ERbeta ligand treatment PNAS, September 11, 2007; 104(37): 14813 - 14818. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R. Thorn, M. J. Meyer, M. E. Van Amburgh, and Y. R. Boisclair Effect of Estrogen on Leptin and Expression of Leptin Receptor Transcripts in Prepubertal Dairy Heifers J Dairy Sci, August 1, 2007; 90(8): 3742 - 3750. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Sasahara, H. Shikimi, S. Haraguchi, H. Sakamoto, S.-i. Honda, N. Harada, and K. Tsutsui Mode of Action and Functional Significance of Estrogen-Inducing Dendritic Growth, Spinogenesis, and Synaptogenesis in the Developing Purkinje Cell J. Neurosci., July 11, 2007; 27(28): 7408 - 7417. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. B. Jelks, R. Wylie, C. L. Floyd, A. K. McAllister, and P. Wise Estradiol Targets Synaptic Proteins to Induce Glutamatergic Synapse Formation in Cultured Hippocampal Neurons: Critical Role of Estrogen Receptor-{alpha} J. Neurosci., June 27, 2007; 27(26): 6903 - 6913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Xue, J. Pamidimukkala, D. B. Lubahn, and M. Hay Estrogen receptor-{alpha} mediates estrogen protection from angiotensin II-induced hypertension in conscious female mice Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1770 - H1776. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Hart, M. A. Snyder, T. Smejkalova, and C. S. Woolley Estrogen Mobilizes a Subset of Estrogen Receptor-{alpha}-Immunoreactive Vesicles in Inhibitory Presynaptic Boutons in Hippocampal CA1 J. Neurosci., February 21, 2007; 27(8): 2102 - 2111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Fan, M. Warner, and J.-A. Gustafsson Estrogen receptor beta expression in the embryonic brain regulates development of calretinin-immunoreactive GABAergic interneurons PNAS, December 19, 2006; 103(51): 19338 - 19343. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. N. Shelley, E. Choleris, M. Kavaliers, and D. W. Pfaff Mechanisms underlying sexual and affiliative behaviors of mice: relation to generalized CNS arousal Soc Cogn Affect Neurosci, December 1, 2006; 1(3): 260 - 270. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Qiu, M. A. Bosch, K. Jamali, C. Xue, M. J. Kelly, and O. K. Ronnekleiv Estrogen Upregulates T-Type Calcium Channels in the Hypothalamus and Pituitary J. Neurosci., October 25, 2006; 26(43): 11072 - 11082. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. H. Morrison, R. D. Brinton, P. J. Schmidt, and A. C. Gore Estrogen, Menopause, and the Aging Brain: How Basic Neuroscience Can Inform Hormone Therapy in Women J. Neurosci., October 11, 2006; 26(41): 10332 - 10348. [Full Text] [PDF]
|
|
|
|
|
|
P. Haeger, M. E. Andres, M. I. Forray, C. Daza, S. Araneda, and K. Gysling Estrogen receptors alpha and beta differentially regulate the transcriptional activity of the Urocortin gene. J. Neurosci., May 3, 2006; 26(18): 4908 - 4916. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Vegeto, S. Belcredito, S. Ghisletti, C. Meda, S. Etteri, and A. Maggi The Endogenous Estrogen Status Regulates Microglia Reactivity in Animal Models of Neuroinflammation Endocrinology, May 1, 2006; 147(5): 2263 - 2272. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Clegg, L. M. Brown, S. C. Woods, and S. C. Benoit Gonadal hormones determine sensitivity to central leptin and insulin. Diabetes, April 1, 2006; 55(4): 978 - 987. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. V. V. Helena, M. de Oliveira Poletini, G. L. Sanvitto, S. Hayashi, C. R. Franci, and J. A. Anselmo-Franci Changes in {alpha}-estradiol receptor and progesterone receptor expression in the locus coeruleus and preoptic area throughout the rat estrous cycle J. Endocrinol., February 1, 2006; 188(2): 155 - 165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Bodo, A. E. Kudwa, and E. F. Rissman Both Estrogen Receptor-{alpha} and -{beta} Are Required for Sexual Differentiation of the Anteroventral Periventricular Area in Mice Endocrinology, January 1, 2006; 147(1): 415 - 420. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Zsarnovszky, H. H. Le, H.-S. Wang, and S. M. Belcher Ontogeny of Rapid Estrogen-Mediated Extracellular Signal-Regulated Kinase Signaling in the Rat Cerebellar Cortex: Potent Nongenomic Agonist and Endocrine Disrupting Activity of the Xenoestrogen Bisphenol A Endocrinology, December 1, 2005; 146(12): 5388 - 5396. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Imamov, G.-J. Shim, M. Warner, and J.-A. Gustafsson Estrogen Receptor beta in Health and Disease Biol Reprod, November 1, 2005; 73(5): 866 - 871. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. Smith, M. J. Cunningham, E. F. Rissman, D. K Clifton, and R. A. Steiner Regulation of Kiss1 Gene Expression in the Brain of the Female Mouse Endocrinology, September 1, 2005; 146(9): 3686 - 3692. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. R. Miller, T. Jover, H. W. Cohen, R. S. Zukin, and A. M. Etgen Estrogen Can Act via Estrogen Receptor {alpha} and {beta} to Protect Hippocampal Neurons against Global Ischemia-Induced Cell Death Endocrinology, July 1, 2005; 146(7): 3070 - 3079. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. Smith, H. M. Dungan, E. A. Stoll, M. L. Gottsch, R. E. Braun, S. M. Eacker, D. K Clifton, and R. A. Steiner Differential Regulation of KiSS-1 mRNA Expression by Sex Steroids in the Brain of the Male Mouse Endocrinology, July 1, 2005; 146(7): 2976 - 2984. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. F. Koehler, L. A. Helguero, L.-A. Haldosen, M. Warner, and J.-A. Gustafsson Reflections on the Discovery and Significance of Estrogen Receptor {beta} Endocr. Rev., May 1, 2005; 26(3): 465 - 478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.B Hawkins, J Godwin, D Crews, and P Thomas The distributions of the duplicate oestrogen receptors ER-{beta}a and ER-{beta}b in the forebrain of the Atlantic croaker (Micropogonias undulatus): evidence for subfunctionalization after gene duplication Proc R Soc B, March 22, 2005; 272(1563): 633 - 641. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Amin, T. Canli, and C. N. Epperson Effect of Estrogen-Serotonin Interactions on Mood and Cognition Behav Cogn Neurosci Rev, March 1, 2005; 4(1): 43 - 58. [Abstract] [PDF]
|
|
|
|
|
|
J. Pamidimukkala, B. Xue, L. G. Newton, D. B. Lubahn, and M. Hay Estrogen receptor-{alpha} mediates estrogen facilitation of baroreflex heart rate responses in conscious mice Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1063 - H1070. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. R. Chakraborty, G. Rajendren, and A. C. Gore Expression of Estrogen Receptor {alpha} in the Anteroventral Periventricular Nucleus of Hypogonadal Mice Experimental Biology and Medicine, January 1, 2005; 230(1): 49 - 56. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. J. S. Miller, S. Suzuki, L. K. Miller, R. Handa, and R. M. Uht Estrogen Receptor (ER){beta} Isoforms Rather Than ER{alpha} Regulate Corticotropin-Releasing Hormone Promoter Activity through an Alternate Pathway J. Neurosci., November 24, 2004; 24(47): 10628 - 10635. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Chakraborty and A. C. Gore Aging-Related Changes in Ovarian Hormones, Their Receptors, and Neuroendocrine Function Experimental Biology and Medicine, November 1, 2004; 229(10): 977 - 987. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. Kudwa, J.-A. Gustafsson, and E. F. Rissman Estrogen Receptor {beta} Modulates Estradiol Induction of Progestin Receptor Immunoreactivity in Male, But Not in Female, Mouse Medial Preoptic Area Endocrinology, October 1, 2004; 145(10): 4500 - 4506. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. M. Abraham, M. G. Todman, K. S. Korach, and A. E. Herbison Critical in Vivo Roles for Classical Estrogen Receptors in Rapid Estrogen Actions on Intracellular Signaling in Mouse Brain Endocrinology, July 1, 2004; 145(7): 3055 - 3061. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Q. Chen, M. Delannoy, C. Cooke, and J. D. Yager Mitochondrial localization of ER{alpha} and ER{beta} in human MCF7 cells Am J Physiol Endocrinol Metab, June 1, 2004; 286(6): E1011 - E1022. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. K. Jezierski and F. Sohrabji Estrogen Enhances Retrograde Transport of Brain-Derived Neurotrophic Factor in the Rodent Forebrain Endocrinology, November 1, 2003; 144(11): 5022 - 5029. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ikeda, A. Nagai, M.-A. Ikeda, and S. Hayashi Sexually Dimorphic and Estrogen-Dependent Expression of Estrogen Receptor {beta} in the Ventromedial Hypothalamus during Rat Postnatal Development Endocrinology, November 1, 2003; 144(11): 5098 - 5104. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. A. Molenda, C. P. Kilts, R. L. Allen, and M. J. Tetel Nuclear Receptor Coactivator Function in Reproductive Physiology and Behavior Biol Reprod, November 1, 2003; 69(5): 1449 - 1457. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
