Regional, Laminar, and Cellular Distribution of Immunoreactivity for ER{alpha} and ERß in the Cerebral Cortex of Hormonally Intact, Adult Male and Female Rats

M.F. Kritzer

Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, NY 11794-5230, USA

Mary Kritzer, Department of Neurobiology and Behavior, SUNY at Stony Brook, Stony Brook, NY 11794-5230, USA. Email: mkritzer{at}notes1.cc.sunysb.edu.


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Summary and Conclusions
 Notes
 References
 
Behavioral, biochemical and anatomical studies suggest that estrogen stimulates structure and/or function in the adult cerebral cortex. The studies presented here used immunocytochemistry to map the {alpha} and ß isoforms of intracellular estrogen receptors (ER{alpha}, ERß) in major subdivisions of adult rat cortex to identify potential sites for relevant receptor-mediated hormone actions. These studies revealed that immunoreactivity for ER{alpha} (ER{alpha}-IR) and ERß (ERß-IR) was present in most cortical areas, was associated exclusively with neurons, and was similar in males and females. Each receptor isoform also had its own unique distribution with respect to cortical regions, layers, and cells. In sensorimotor areas, for example, ERß-IR was more prominent than ER{alpha}-IR, and was concentrated in layer V neurons that were immunoreactive for parvalbumin. In contrast, ER{alpha}-IR was scattered among parvalbumin-immunonegative cells in layers II/III and V/VI. Likewise, in entorhinal cortex, ERß-IR was present in calbindin-containing cells in layers III–VI, while ER{alpha}-IR was restricted to small numbers of calbindin-negative neurons in infragranular layers. In sum, ERß-IR and ER{alpha}-IR were differentially distributed both with respect to cortical compartments and with respect to each other. Accordingly, estrogen activation at these two sites may be anticipated to impact disparate sets of cortical circuits, cells, and functions.


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Summary and Conclusions
 Notes
 References
 
The cerebral cortex is a recognized target of ovarian hormone stimulation. In addition to developmental influences [e.g. see (Miranda and Toran-Allerand, 1992Go; Lustig et al., 1993Go; Stewart and Rajabi, 1994Go], behavioral (Broverman et al., 1981Go; Hampson, 1990Go), biochemical (Fink et al., 1996Go), and anatomical studies in humans and/or other mammals (Kritzer and Kohama, 1998Go,1999Go; Kritzer, 2000Go) suggest that estrogen also stimulates the mature cerebrum. Among the mechanisms that could mediate this stimulation are intracellular receptor–hormone interactions. In the adult cortex in particular these could involve estrogen activation of the classical or {alpha} isoform of the ER (ER{alpha}) and/or the more recently identified ß isoform of the ER [ERß, see Kuiper et al. (Kuiper et al., 1996Go)], both of which seem to be present in the cortices of sexually mature animals (Li et al., 1997Go; Shughrue et al., 1997Go; Weiland et al., 1997Go; Mufson et al., 1999Go). There are hints, however, that the functional roles of ER{alpha}-vs ERß-mediated processes in the adult cerebrum may be very different. Both immunocytochemical and in situ hybridization studies, for example, indicate that the two receptor isoforms differ substantially in their relative abundance (Simerly et al., 1990Go; Li et al., 1997Go; Shughrue et al., 1997Go; Mufson et al., 1999Go), and in the cortical compartments within which they are localized (Shughrue et al., 1997Go). Because ligand activation at ER{alpha} vs ERß sites can differentially induce morphological endpoints (Patrone et al., 2000Go) and gene transcription at, for example, AP-1 sites (Paech et al., 1997Go), a precise knowledge of the cortical localization of each receptor subtype is needed to evolve a complete understanding of how estrogen impacts cortical function and form. Because ER{alpha} and ERß subunits can also form heterodimers that are functionally, i.e. transcriptionally, distinct from both ER{alpha} and ERß homodimers (Pettersson et al., 1997Go; Ogawa et al., 1998Go), it may be equally important to determine whether and where in the cortex these two signaling pathways intersect. The studies presented here addressed each of these issues by using immunocytochemistry to generate detailed maps of the distributions of ER{alpha} and ERß proteins in the cerebral cortices of hormonally intact adult male and female rats. Specifically, patterns of immunoreactivity for the ER{alpha} and ERß receptor isoforms were each defined in relation to cytoarchitectonically identified regions and layers of all major subdivisions of the rat cerebral cortex. In these areas as well, both receptor isoforms were charted in relation to individual cells identified by cytoplasmic markers including glial fibrillary acidic protein, neuron-specific enolase, and the calcium binding proteins parvalbumin (PV) and calbindin (CB). Together these analyses provided a comprehensive, fine-grained definition of the distributions of ER{alpha} and ERß in the adult rat cortex. Consideration of the known functional specializations of the particular cortical regions, layers, and cells that immunoreactivity for each receptor isoform occupied suggests that ER{alpha}-and ERß-mediated pathways may indeed be poised to influence different cells and circuits in the adult rat cerebral cortex.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Summary and Conclusions
 Notes
 References
 
Tissue

A total of 18 adult Sprague–Dawley rats (12 males, 6 females) were used. All animals were hormonally intact adults weighing between 200 and 400 g. Vaginal cytology smears were taken to track the estrous cycles of all females prior to sacrifice, and were also used to verify which animals were in proestrus (n = 3) and which were in diestrus (n = 3) at the time of euthanasia.

Tissue Preparation

Rats were deeply anesthetized with a mixture of ketamine and xylazine (90 and 10 mg/kg, respectively) delivered intramuscularly. After behavioral checks were made to ensure the disappearance of deep reflexes, animals were transcardially perfused with 100–200 ml of phosphate-buffered saline flush, followed by 500–1000 ml of one of the following fixatives: 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4 (two males); 4% paraformaldehyde containing 15% saturated picric acid in 0.1 M PB, pH 7.4 (two males); 2% paraformaldehyde in 0.1 M PB, pH 7.4 (two males); 2% paraformaldehyde containing 15% saturated picric acid in 0.1 M PB, pH 7.4 (four males, six females); 1% paraformaldehyde in 0.1 M PB, pH 7.4 (one male); 1% paraformaldehyde containing 15% saturated picric acid in 0.1 M PB, pH 7.4 (one male). Following perfusion, brains were removed and placed in 0.1 M PB containing 10%, 20%, and finally 30% sucrose for cryoprotection. After brains had sunk in the most concentrated sucrose solution, they were rapidly frozen in powdered dry ice, secured to a freezing microtome stage, and serially sectioned (40 µm) from the anterior to the posterior pole.

Histology

Single Label Immunohistochemistry

For studies using only one antibody (anti-ERß or anti-ER{alpha}), sections were first rinsed in 0.1 M PB, pH 7.4, reacted with 1% H2O2 in PB for 45 min, rinsed again in 0.1 M PB, and were then incubated in 1% sodium borohydride in PB (45 min). Sections were then rinsed in 50 mM tris buffered saline (TBS), pH 7.4, and placed in blocking solution [TBS containing 10% normal swine serum (NSS) for 2 h at room temperature]. Sections were then incubated in primary antisera, diluted in TBS containing 1% NSS, for 3 days at 4°C. After this incubation, sections were rinsed in TBS and then placed in biotinylated secondary antibodies (2 h, room temperature, working dilution 1:100). Sections were again rinsed in TBS, and incubated in avidin–biotin-complexed horseradish peroxidase (ABC, Vector Labs, Burlingame, CA; 2 h, room temperature). After additional rinses in TBS and then in tris buffer (TB), pH 8.0, sections were reacted using 0.07% 3,3'-diaminobenzadine (DAB) and 0.06% nickel ammonium sulfate as chromagen.

Double-label Immunohistochemistry

For double-labeling experiments, antigens were immunolabeled sequentially using a first primary antibody generated in rabbit (polyclonal) and a second generated in mouse (monoclonal). Labeling of the first antibody utilized the protocol for single-label immunohistochemistry described above; in every case, the first antibodies used were polyclonals recognizing the intracellular receptors and the chromagen used was DAB/nickel. After the first reaction was completed, sections were rinsed in TBS, placed in blocking solution (TBS containing 10% NSS, 2 h), and then incubated in a next monoclonal primary antibody (1 day, 4°C). After incubations with biotinylated secondary antibodies and the ABC solution, sections were rinsed first in TBS, and then in TB, pH 7.6. Sections were then reacted for a second time using DAB alone as chromagen. All immunoreacted sections (single- or double-label), were slide-mounted, dehydrated and placed under coverslips; some sections were also lightly counterstained for Nissl substance by using 1% cresyl violet prior to coverslipping.

Antibodies

Polyclonal antibodies were used that selectively recognize the {alpha} and ß isoform of the intracellular estrogen receptor [{alpha} isoform (Upstate Biotechnology, Lake Placid, NY, lot no. 18389), ß isoform (Zymed Laboratories, South San Francisco, CA, lot no. 01162952)]. The specificity of both antibodies has been tested on Western blots, and neither was pre-absorbed prior to use [see also Shughrue and Merchenthaler (Shughrue and Merchenthaler, 2001Go)]. The anti-ER{alpha} was used at a working dilution of 1:6000, and the ERß antibody was used at a dilution of 1:750. Monoclonal antibodies recognizing neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were purchased from Chemicon International Inc. (Temecula, CA), and were used at working dilutions of 1:100 and 1:300, respectively. The oligodendrocyte-specific monoclonal antibody (RIP) that was used was developed by Dr Susan Hockfield (Yale University, New Haven, CT) and was obtained from the Developmental Studies Hybridoma Bank, a facility maintained by the Department of Biological Sciences, University of Iowa, Iowa City, IA, under contract NO1-HD-7-3263 from the National Institute of Child Health and Development (NICHD); this antibody was used at a working dilution of 1:3. Monoclonal antibodies recognizing parvalbumin (PV) or calbindin (CB) were purchased from Sigma Chemical Corp. (St Louis, MO); both were used at working dilutions of 1:1000.

Control Experiments

The immunohistochemical labeling protocols (single and double) described above were carried out on representative sections from male and female animals with the omission of primary or secondary antibodies.

Data Analysis

Sections of the rat cerebrum analyzed were taken at 400 µm intervals from the rostral to caudal poles. In most cases cytoarchitecture was defined according to Zilles and Wree, in counterstained sections (Zilles and Wree, 1985Go). Any areas that were defined by other criteria are so identified in the text.

Qualitative Analysis

Detailed qualitative assessments of labeling were carried out in all major cytoarchitectonic divisions of the rat cerebral cortex. In each area, assessments were made of the appearance, regional and laminar distribution, relative abundance and degree of colocalization of ER{alpha}-IR and ERß-IR with NSE, GFAP, RIP, PV and CB.

Quantitative Estimates

Counts of the number of ERß-IR cells present per unit area were made in representative sensory, motor and association cortical areas. The regions selected for analysis were areas Cg2, Fr2, Fr1, Par1, AID and AIV, with evaluations taking place on sections taken roughly to levels 2.2–2.7 mm rostral to bregma as per the rat atlas of Paxinos and Watson (Paxinos and Watson, 1986Go). Immunoreactive nuclei were counted on a Zeiss Axioskop (Carl Zeiss Inc., Thornwood, NY) under brightfield illumination using a 20x objective and calibrated eyepiece reticule (200 x 200 µm) to delineate counting boxes. For each area evaluated, the counting box was placed within the region's layer V, and counts were made of all immunoreactive nuclei lying within the box, using the top and right sides of the grid as exclusion lines. This process was repeated per area for three separate sections from each animal subject, with measures summed to produce group means. These cell counts were not corrected for tissue shrinkage, or for potential group differences in average cell size or overall cell density. Because measures did not rigorously adhere to stereological principles, these data were used only for comparative purposes in the substantiating visual impressions.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Summary and Conclusions
 Notes
 References
 
The studies presented here describe the distribution of estrogen receptor-immunoreactivity in identified regions, layers, and cells of all major cytoarchitectonic divisions of the iso- and allocortex in hormonally intact adult male rats, and in adult female rats that were in either in proestrous or diestrous at the time of euthanasia. In no cases were obvious sex differences or estrus cycle-dependent variations in the appearance, distribution, or apparent density of receptor immunoreactivity observed. Accordingly, all of the data presented should be assumed to apply equally to all three animal groups evaluated.

Antibody Specificity

Commercially available antibodies were used in single- and double-label immunocytochemical studies to localize and colocalize the {alpha} and ß isoforms of the intracellular estrogen receptor (ER{alpha}, ERß). In sections labeled with the anti-estrogen receptor antibodies alone, immunoreactive (IR) profiles corresponded to small spots of blue/black DAB/nickel reaction product that were localized mainly to cell nuclei (Fig. 1Go). The gross distributions of these nuclei in the cerebral and cerebellar cortices, as well as in subcortical structures such as the amygdala and hypothalamus were good matches for previous descriptions of receptor proteins mapped using immunocytochemistry (Li et al., 1997Go; Weiland et al., 1997Go; Mufson et al., 1999Go), and/or for receptor mRNAs mapped using in situ hybridization (Simerly et al., 1990Go; Shughrue et al., 1997Go). For example, ER{alpha}-IR and ERß-IR cells were both prominent in medial aspects of the amygdala, ER{alpha}-IR cells clearly outnumbered ERß-IR cells in the peri-aqueductal gray, and ERß-IR profiles predominated in the cerebellar and cerebral cortices. Immunolabeling for the five cytoplasmic markers used also gave rise to expected patterns of staining, with labeled cortical cells displaying morphologies typical of oligodendrocytes (anti-RIP, Fig. 2BGo), astrocytes (anti-GFAP, Fig. 2A,CGo), or neurons (anti-NSE, anti-PV, anti-CB, see Fig. 2Go). No patterned or cellularly specific labeling was evident, however, in control experiments in which the primary or secondary antibodies were omitted from immunolabeling protocols, further supporting the specificity of the antibodies used for marking intended antigens.



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Figure 1. Representative high power photomicrographs showing immunoreactivity for the {alpha} and ß isoforms of the intracellular estrogen receptor (ER{alpha}, ERß) taken from layer V of the primary somatosensory cortex of a hormonally intact adult male rat. Immunoreactivity for both receptor types is concentrated over small nuclei that are usually round (A, B) but sometimes are more fusiform in shape (C, D). In some cases, patchy immunoreactivity for ER{alpha} can be seen extending into the cytoplasm and proximal processes of cell somata (A, D). Scale bar = 10 µm.

 


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Figure 2. Representative color photomicrographs illustrating the anatomical relationships between nuclei immunoreactive for ER{alpha} or ERß, and immunoreactivity for GFAP (A, C), the oligodendrocyte marker RIP (B), NSE (DH), PV (I, J, L, N, O) or CB (K, M). Nuclei that were immunoreactive for ERß or ER{alpha} were never found in cells expressing glial markers (AC). Almost all receptor-positive nuclei were, however, sequestered within cells that were immunoreactive for NSE (DH). Large and medium-sized pyramidal cells did not contain receptor-positive nuclei (e.g. D), but cells immunoreactive for ER{alpha} in deeper cortical layers did have vaguely pyramidal form (e.g. H). Cells that were immunoreactive for ERßNSE had bipolar (G), multipolar (E), or more irregular morphologies (F) that did not resemble pyramidal cells. Most of the ERß-immunoreactive cells were also immunoreactive for PV (I, J, L, N). Neurons that were immunoreactive for ER{alpha} were almost always immunonegative for CB (M) and PV (O). Thus, in areas such as AID (N, O) where ER{alpha} and ERß spatially overlapped, the two receptor isoforms appeared to be found in different cells populations. All sections shown are from hormonally intact adult male rats; unless otherwise indicated (N, O) sections are from primary motor or primary somatosensory cortex. Scale bars: K, M, N, O = 20 µm; all others = 10 µm.

 
Sensitivity of ER-IR to Fixation

A number of earlier studies using immunocytochemistry to examine the distributions of ER{alpha} or ERß in rat brain used animals that were perfused with fixatives containing acrolein [e.g. Weiland et al. and Jakab et al. (Weiland et al., 1997Go; Jakab et al., 2001Go)]. Initial experiments of this study tested whether appropriate signal-to-noise ratios of ER-IR could be obtained using alternatives to this fixative. The fixatives tested included paraformaldehyde solutions of varying strengths, as well as paraformaldehyde fixatives augmented with 15% saturated picric acid. These comparative studies revealed sensitivity of the anti-ERß antibodies to conditions of fixation. Whereas immunolabeling for all of the other antibodies used were similar under all fixation regimens, immunoreactivity for ERß (ERß-IR) was markedly attenuated in brains fixed with 4% paraformaldehyde (with or without picric acid), and was considerably stronger in the more dilute aldehyde fixes. Among these, fixes combining the 15% picric acid solution with low paraformaldehyde concentrations (1 or 2%) consistently gave the strongest ERß-immunoreactive signal along with almost undetectable levels of staining in surrounding tissue space. In choosing between the 1% paraformaldehyde–15% picric acid and the 2% paraformaldehyde–15% picric acid fixatives, which gave similar signal-to-noise ratios, the latter was selected as more strongly fixed tissue better withstand the protracted processing involved in double-label immunocytochemistry.

Identification of Cellular Phenotype of Cortical ER-IR Profiles

Both ER{alpha}-IR and ERß-IR cells were distinguished by strong to moderate levels of immunolabeling that was concentrated over their nuclei (Fig. 1Go). Most labeled nuclei were round in shape and small to medium-sized (5–10 µm) in diameter (Fig. 1A,BGo). For both ER{alpha} and ERß as well, occasional immunoreactive cells were also found that had more irregularly, e.g. fusiform, shaped nuclei (Fig. 1C,DGo). There were, however, subtle features of labeling that distinguished ERß-IR from ER{alpha}-IR cells. Most notably, in ERß-IR cells the DAB/nickel reaction product stood out sharply over nuclear compartments, but usually did not rise above background levels in the surrounding cytoplasm (Fig. 1B,CGo). In ER{alpha}-IR cells, on the other hand, labeled nuclei were often surrounded by cytoplasm that displayed weak, often patchy immunoreactivity that in some cases revealed enough morphology to suggest that the cells were neurons (Fig. 1AGo). For the most part, however, it was impossible to reliably classify cells as neuronal or glial based solely on labeling for ER{alpha} or ERß alone. For this purpose, double-label immunocytochemistry experiments pairing ER-IR with immunoreactivity for NSE, GFAP, and the oligodendrocyte marker RIP were performed. These analyses revealed no ER-IR nuclei that were co-labeled with the glial markers (anti-GFAP or anti-RIP, Fig. 2ACGo). However, nearly every ER-IR ({alpha} and ß) nucleus encountered was surrounded by cytoplasm that was immunoreactive for NSE (Fig. 2DHGo). For ER{alpha}-IR cells, the concomitant NSE labeling usually revealed neurons of two basic types. Nearer the pial surface, ER{alpha}-IR was typically found in neurons with NSE-labeled somata that were small and round and that emitted few obvious processes. Deeper within the cortex, however, ER{alpha}-IR was localized almost exclusively in small neurons with somata that were more triangularly shaped and that gave rise to single, striking apical processes (Fig. 2HGo). ERß-IR, on the other hand, was localized in small NSE-IR cells that were most often either fusiform (Fig. 2F,GGo) multipolar, or more complex or irregular in shape (Fig. 2EGo). A final series of double-labeling experiments using antibodies recognizing the calcium binding proteins PV and CB revealed that only in exceptional cases were ER{alpha}-IR neurons also PV-IR or CB-IR. In contrast, the majority of the ERß-IR neurons were PV-IR (Fig. 2I,J,L,NGo) and a few were CB-IR as well. As described separately by receptor subtype below, however, there was some degree of regional and laminar selectivity in the neurochemical signatures of cortical ER-IR cells.

Distribution of ERß in the Cerebral Cortex

A comprehensive survey of the adult rat cerebral cortex revealed that only the transitional zone lying between the supragenual cingulate and granular retrosplenial cortices [see Van Eden and Uylings (Van Eden and Uylings, 1985Go)], was immunonegative for ERß. In every animal examined this field alone stood out as uniquely lacking in receptor-bearing cells (Fig. 3Go). In all other major cytoarchitectonic division of the iso- and allocortex, modest to moderate complements of ERß-IR cells were found. Further, for most regions, ERß-IR was most prominent in layer V. The exceptions to this were the piriform and entorhinal cortices that are discussed in separate sections below.



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Figure 3. Low power photomicrograph showing a lack of immunoreactivity for ERß in the transitional cortical zone as defined by Van Eden and Uylings (Van Eden and Uylings, 1985Go). Labeled nuclei are seen in layer V of the more medially located transitional zone. This area was unique among isocortical and allocortical areas alike in having no nuclei that were immunoreactive for ERß in any of the animals examined. The tissue section shown is from a hormonally intact adult male rat. Abbreviation: cc = corpus callosum. Scale bar = 200 µm.

 
ERß in Sensorimotor and Association Areas of the Cerebral Cortex

The majority of sensory, motor, and association areas of the rat cerebrum displayed ERß-IR that was most prominent in layer V. However, differences in the apparent density and/or in the laminar, tangential, or cellular distribution of receptor-bearing nuclei distinguished the association from the sensorimotor areas, and further distinguished some of the individual fields examined within both these two broad categories.

Association Areas: Regional and Laminar Distribution

The association areas of the cerebrum examined included cingulate, insular, and retrosplenial fields. Among these, the medial prefrontal (Cg1–3), retrosplenial (RSG and RSA), and ventral anterior insular (AIV) areas were each characterized by sparse complements of ERß-IR cells that were distributed evenly and exclusively among the sublayers of lamina V (e.g. Fig. 4ACGo). In the dorsal and posterior insular cortices (AID, AIP), receptor-positive cells were also restricted to this same infragranular layer. However, in these regions ERß-IR cells were much more numerous than in other association areas, and seemed to be more organized into clusters (compare Fig. 4A,B,CGo). Thus, in contrast to the scatter of ERß-IR cells in, for example, area Cg1, receptor-bearing neurons in areas AID and AIP were often found arrayed in groups of three to five cells that overall had densities (per unit area) that were some three times higher than those of ERß-IR cells in the cingulate areas.



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Figure 4. Digitized camera lucida drawings of the distributions of cells that are immunoreactive for PV (plusses), nuclei that are immunoreactive for ERß (open circles) and cells that were both PV- and ERß-immunoreactive (filled circles) in representative regions of isocortex. In all areas, ERß-immunoreactive nuclei are most abundant in layer V; for the association areas (AC), immunoreactive nuclei are only found in this layer. In sensory (D) and motor (E, F) areas, small numbers of immunoreactive nuclei are also found outside of layer V. In all areas as well, most cells immunoreactive for ERß were also immunoreactive for PV; in the association areas all ERß-immunoreactive cells contained PV, but in sensorimotor areas (DF), small numbers of cells were also found that were immunoreactive for ERß but immunonegative to PV. All drawings shown represent tissue sections from hormonally intact adult male rats. Roman numerals on the right side of each drawing identify cortical layers. Abbreviation: wm = white matter.

 
Association Areas: Cellular Distribution

Double-labeling studies using receptor and calcium binding protein antibodies showed that, without exception, all of the ERß-IR cells present in the association cortices were PV-IR (Fig. 4ACGo). In most areas, these ERß/PV-IR cells included equally prevalent examples of larger multipolar and smaller, morphologically simpler (e.g. bipolar) neurons. In areas AID and AIP, however, the ERß/PV-IR cells corresponded almost exclusively to the larger, multipolar types of cells (Fig. 2NGo). Finally, all the association areas shared a modest complement of ERß-IR cells lying mainly in the lower half of layer V that were also CB-IR (Fig. 5CGo). Because the ERß-IR cells identified in alternate sections were all PV-IR, it was assumed that these ERß/CB-IR cells represent subsets of receptor-bearing neurons that contained both calcium binding proteins.



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Figure 5. Digitized camera lucida drawings of the distributions of cells immunoreactive for CB (plusses), nuclei that are immunoreactive for ERß (open circles) and cells that were both CB- and ERß-immunoreactive (filled circles) in representative regions of sensory, motor and association areas of the cortex. In contrast to double labeling for PV, only small populations of ERß-immunoreactive cells contained CB. In the association areas such as area Cg1 (C), the few ERß-/CB-immunoreactive cells that were present were typically found in the lower half of layer V. In sensorimotor areas including the primary somatosensory (A, Par1) and primary motor cortex (B, Fr1), nuclei that are immunoreactive for both ERß and CB were more broadly scattered. All drawings shown represent tissue sections from hormonally intact adult male rats. Superficially placed asterisks mark strata where dense populations of cells weakly immunoreactive for CB are found, but for the sake of clarity are not represented in the figure. Roman numerals on the right side of each drawing identify cortical layers. Abbreviation: wm = white matter.

 
Sensorimotor Areas: Regional and Laminar Distribution

The sensorimotor areas of the cortex examined included the primary and secondary somatosensory (Par1, Par 2), auditory (Te1, Te3), visual (Oc1, Oc2), and motor (Fr1, Fr2) areas. Each of these regions shared a basic pattern of receptor immunoreactivity that was different in several respects from that seen in the association areas. First, although labeled cells were most conspicuous in layer V, the sensorimotor fields also had ERß-IR cells located outside this layer. Thus, in every region examined there were moderate numbers of neurons seen in layer I, with smaller numbers of receptor-positive neurons also found in layers II/III and VI (Fig. 4DFGo). The apparent density of ERß-IR cells within layer V of the sensorimotor regions was also considerably higher than in the association areas (compare Figs 4B,Cand 4DFGo). Quantitative estimates of the areal densities of ERß-IR cells in representative areas of the frontal cortex bore this out. In the primary motor and somatosensory fields, for example, cell densities were on the order of three times higher than values for cingulate and insular areas.

Although immunoreactivity in all sensorimotor fields was similar, regional variations in labeling could also be appreciated. In many cases, these variations seemed to correspond to differences in the cytoarchitecture of layer V. For example, areas such as primary visual cortex where layer V is relatively compact had immunoreactivity that was focused into bands that were narrower than in, for example, motor areas where layer V is broader (Figs 4DFGo). The somatosensory cortices, however, had more strikingly different patterns of immunolabeling. Specifically, whereas labeling in most areas of cortical zones was evenly distributed among the sublaminae of layer V, in areas Par1 and Par2 ERß-IR was more restricted to the lower half of layer V, i.e. sublayer Vb (Fig. 6Go).



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Figure 6. Low power photomicrographs showing the distribution of nuclei immunoreactive for ERß in layer V of the primary motor (A, area Fr1) and primary somatosensory cortex (B, area Par1). Both panels are from a hormonally intact adult male rat, and have been counterstained with 1% cresyl violet to reveal cortical layers (identified by roman numerals, right). In motor cortex, immunoreactive nuclei are found throughout the depths of layer V; in somatosensory cortex, nuclei are confined largely to the lower half of layer V. A similar confinement of ERß-immunoreactive nuclei to layer Vb was also seen in the secondary somatosensory area. Scale bar = 250 µm.

 
Sensorimotor Areas: Cellular Distribution

In addition to laminar differences, ERß-IR in the sensorimotor versus association areas was also distinguished by differences in the populations of neurons that were receptor-bearing. Specifically, whereas all of the ERß-IR cells in the association cortices were PV-IR, it was only most of the ERß-IR cells in the sensorimotor areas that contained this calcium binding protein. Thus, in every region evaluated, between 10 and 20% of the ERß-IR cells in layer V, and roughly half of the ERß-IR cells found outside of this layer were PV-immunonegative (Fig. 4DFGo). As in the association areas, a small number of ERß-IR cells that were CB-IR were also observed (Fig. 5A,BGo). While such cells were most common in layer Vb in the association areas, in the sensorimotor fields, they could be found throughout layers II/III and V/VI. Numerically, these ERß/CB-IR cells did not seem sufficient to account for all ERß-IR cells left unlabeled by PV in alternate sections.

ERß in Piriform and Entorhinal Cortex: Regional, Laminar and Cellular

In contrast to the sometimes subtle distinctions that differentiated receptor immunoreactivity among some of the sensory, motor and association regions of the cortex, more noticeable differences in the laminar and cellular distribution of ERß-IR set off the piriform and entorhinal cortices both from isocortical areas and from one another. For example, while most cortical labeling was concentrated in layer V, in the three-layered piriform cortex ERß-IR was localized to its deepest layer (layer III, Fig. 7AGo), while in the entorhinal cortices, ERß-IR was more evenly scattered across layers III–VI (Fig. 7CGo). Further, while ERß-IR cells in sensorimotor and association areas were predominantly and in some areas exclusively PV-IR, in the piriform cortex typically less than half of the ERß-IR cells present within a given field were PV-IR, and in the entorhinal cortices the proportion of ERß/PV-IR cells dropped from ~50% in the lateral entorhinal area to almost zero in the medial entorhinal cortex (Fig. 7CGo). Many of the ERß-IR cells in these regions were, however, immunoreactive for CB. Thus, in the piriform cortex, as many as two-thirds of the ERß-IR cells present were also CB-IR (Fig. 7BGo), and in the entorhinal areas almost all visible receptor-bearing cells were also CB-IR (Fig. 7DGo).



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Figure 7. Digitized camera lucida drawings of the distributions of cells immunoreactive for ERß (A, C open circles; B, D open squares); ERß and PV (A, C filled circles); ERß and CB (B, D filled squares); and immunoreactive for CB or PV alone (plusses) in piriform (PIR; A, B) and entorhinal cortex (ENT; C, D). In piriform cortex, receptor-immunoreactive cells were found mainly in layer III; about a third of these were immunoreactive for PV (A), and roughly two-thirds were immunoreactive for CB (B). In the entorhinal cortex, ERß-immunoreactive cells were distributed in layers III–VI; in lateral portions of this area, roughly half of ERß-positive cells were immunoreactive for PV (C) and half were immunoreactive for CB (D). In the medial entorhinal cortex, the proportion of ERß-immunoreactive cells that contained PV diminished, and the proportion of cells containing CB rose. All drawings shown represent tissue sections from hormonally intact adult male rats. Roman numerals on the right side of each drawing identify cortical layers. Abbreviation: wm = white matter.

 
ER{alpha} in the Cerebral Cortex

The distribution, apparent density, and neurochemical signatures of ER{alpha}-IR cells in the rat cerebral cortex provided contrasts to corresponding parameters of immunoreactivity for ERß. As a general rule, for example, ER{alpha}-IR cells were noticeably less numerous than ERß-IR cells. Perhaps as a result of their relatively low densities, it was only in exceptional cases that clear cut regional differences in the distribution or density of ER{alpha}-IR cells could be discerned among individual cortical areas. As for ERß-IR, however, the patterns of ER{alpha}-IR in piriform and entorhinal areas were readily distinguished from those in some of the more laterally situated sensorimotor and association areas of the cerebrum. Descriptions of these two areas are thus treated below in separate sections, as they were for ERß-IR.

ER{alpha} in Sensorimotor and Association Areas: Regional, Laminar, and Cellular Distribution

Most sensorimotor and association areas of the cortex had only sparse complements of ER{alpha}-IR cells, which were found mainly in layers II/III and V/VI (Figs 8 and 9BGoGo). Although few in number, there were hints that ER{alpha} cells were not randomly distributed. It was not uncommon, for example, to find a repeated pattern of supragranular ER{alpha} labeling that was characterized by small numbers of receptor-positive cells studding the very top of layer II (Figs 8 and 9A,CGoGo). The granular and agranular insular cortices stood out, however, as areas where ER{alpha}-IR was markedly more dense than in laterally flanking cortical zones, and was more obviously concentrated in layer V (Figs 8 and 9C,DGoGo). These features gave ER{alpha}-IR in these regions a likeness to ERß-IR. Double-labeling studies revealed, however, that whereas all of the ERß-IR cells in these association areas were PV-IR, few to none of the ER{alpha}-IR cells identified here — or indeed in any area of the sensorimotor or association areas examined — were PV-IR (Fig. 2OGo) or CB-IR (Fig. 2MGo).



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Figure 8. Digitized camera lucida drawings of representative brain sections charting the locations of cells immunoreactive for ER{alpha}. Most areas of the cortex had small numbers of receptor-immunoreactive cells scattered in layers II/III and V/VI. A repeated pattern was immunolabeling of a few cells along the border between layers I and II. The allocortical piriform (Pir) and entorhinal (Ent) cortices also had sparse populations of receptor-bearing neurons that were most often found in deepest layers. Receptor immunoreactivity was more dense in the cortex surrounding the rhinal fissure, e.g. in anterior insular (AID, AIP) and perirhinal cortex (PRh). The higher density of receptor-immunoreactive cells and their unique concentration in layer V was reminiscent of the pattern of immunoreactivity for the ß isoform of the estrogen receptor. Other abbreviations: Cg, cingulate cortex; Cd, caudate nucleus; Fr, motor cortex; Hp, hippocampus; Oc, visual cortex; Par, somatosensory cortex; RSG, RSA, granular and agranular retrosplenial cortex; Th, thalamus. All drawings shown represent tissue sections from hormonally intact adult male rats.

 


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Figure 9. Representative low power photomicrographs showing the distribution of nuclei immunoreactive for ER{alpha} in sensory (A, Par1), motor (B, Fr1) and associational areas (C, AID; D, AIV). Many cortical regions displayed a stereotyped pattern of labeled cells along the very top of layer II that is illustrated in panel (A) [arrowheads, see also panel (C)]. In areas AID (C) and AIV (D), ER{alpha}-immunoreactive nuclei were more numerous and uniquely surrounding the rhinal sulcus (see also Fig. 8Go). The tissue sections shown are from hormonally intact adult male rats. Scale bars: A = 100 µm, BD = 250 µm.

 
ER{alpha} in the Piriform and Entorhinal Areas: Regional, Laminar and Cellular

Immunoreactivity for ER{alpha} was sparse in the piriform and entorhinal cortices (Fig. 8Go). In these phylogenetically older regions, ER{alpha}-IR cells were mainly found in their deeper layers, and did not include the stereotyped labeling of cells in the top of layer II that was prominent in the more laterally situated areas, e.g. areas Par1 and Fr1. Thus, in the piriform cortex, ER{alpha}-IR was limited to small numbers of cells that peppered layer III, while in the entorhinal cortices receptor-positive cells were distributed mainly within layers V/VI. Double labeling with anti-ER{alpha} antibodies and anti-CB or anti-PV antibodies identified only one or two cases of colocalization between ER{alpha}-IR and two calcium binding proteins within a given section.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Summary and Conclusions
 Notes
 References
 
Behavioral, biochemical, and anatomical studies suggest that estrogen maintains some ability to stimulate structure and/or function in the cerebral cortex in mature humans and animals. The studies presented here used immunocytochemistry to map the distribution of the {alpha} and ß isoforms of the intracellular estrogen receptor in all major subdivisions of the adult rat cerebral cortex in order to gain insight into potential target sites for relevant receptor-mediated hormone actions. These studies revealed that with only rare exceptions, ER{alpha}-IR and/or ERß-IR cells were present in all major fields of the iso- and allocortex of hormonally intact adult male and female rats. Although both were similarly distributed in males and females, each receptor isoform showed its own unique, highly selective distribution with respect to identified cortical regions, layers, and cells. These distributions were similar to previous immunocytochemical and/or in situ hybridization analyses of receptor subtype-specific proteins and/or mRNAs (Simerly et al., 1990Go; Shughrue et al., 1997Go; Weiland et al., 1997Go; Mufson et al., 1999Go), thus supporting the specificity of the antibodies used to recognize specific estrogen receptor subtypes. The correspondence with in situ hybridization studies also supports the conclusion that previously localized transcripts may be active in producing receptor proteins. There is less compelling correspondence, however, between the distribution of receptive machinery detected by immunocytochemistry or in situ hybridization and the distribution of cortical cells identified by hormone binding or uptake. Although together the summed patterns of ER{alpha}-IR and ERß-IR observed in this study provide an excellent match for the distribution of cells concentrating the synthetic estrogen [3H]11ß-methoxy-17-ethyl estradiol in the cerebral cortex of neonatal rats (Sheridan 1979Go), receptor immunoreactivity appears to be in excess of maps of [3H]-estradiol or [125I]11ß-methoxy-16{alpha}-iodoestradiol concentrating cells generated in the cortices of adult rats and mice (Pfaff and Keiner, 1973Go; Shughrue et al., 1990Go). Although there may be technical reasons for these spatial mismatches, e.g. the specific activity of radiolabeled probes, until resolved some caution may be warranted in interpreting receptor-enriched loci identified by immunocytochemistry and/or in situ hybridization alone as sites of functionally patent hormone–receptor interactions.

ERß-IR and ER{alpha}-IR Does not Colocalize with Macroglial Antigens in the Adult Rat Cerebrum

Astrocytes, oligodendrocytes, and microglia have all been identified as reactive to estrogen stimulation in the cerebral or hippocampal cortices (Day et al., 1993Go; Garcia-Estrada et al., 1993Go) and/or in culture (Garcia-Segura et al., 1995Go). The question of whether and which of these cells are actually estrogen receptor-bearing, however, has only been partially addressed for the cerebral cortex. For example, while previous studies have shown that immunoreactivity for ER{alpha} is not colocalized with the astrocyte marker GFAP in the hippocampus or neocortex (Weiland et al., 1997Go), anatomical relationships between ER{alpha} and other glial cell types, e.g. microglia, oligodendrocytes, have only been explored in culture [e.g. Gudino-Cabrera and Nieto-Sampedro and Mor et al. (Gudino-Cabrera and Nieto-Sampedro, 1999Go; Mor et al., 1999Go)]. Previous investigations of ERß–glial relationships are also limited and to date include only analyses of ERß-IR and GFAP-IR colocalization in the hippocampus and cerebellum (Azcoitia et al., 1999Go; Jakab et al., 2001Go). The studies presented here are thus among the first to comprehensively examine the co-distribution of immunoreactivity for ER{alpha}, ERß, oligodendrocytes, and astrocytes in major divisions of the iso- and allocortex. None of these combinations, however, revealed evidence for colocalization between receptor and glial antigens within single cells. It is possible that cortical cells do exist that contain markers for both ER and glia, but which go undetected — perhaps due to low antigen levels. However, this possibility seems remote since the dimensions of cortical ER{alpha}-IR and ERß-IR nuclei were typically larger than the nuclear compartments visible in immunolabeled astrocytes and oligodendrocytes. Although not tested directly, similar arguments suggest that colocalization of ER{alpha} and/or ERß in cortical microglia may be even less likely, as these cells are smaller still than the two macroglial populations examined. Thus, while glial cells in the cerebral cortex may be sensitive to estrogen stimulation, the data presented here and previously suggest that observed effects may be indirect and/or perhaps mediated by receptors other than ER{alpha} or ERß sites.

ER{alpha}-IR and ERß-IR is Present in Different Subsets of Neurons in the Adult Rat Cerebrum

The negative findings obtained with respect to glial cell markers stood in sharp contrast to positive findings of colocalization between ER-IR and immunoreactivity for NSE that surrounded almost every receptor-positive nucleus examined. While previous studies have alluded to the neuronal nature of ER-bearing cortical cells based either on morphology (Pfaff and Keiner, 1973Go) or by exclusion from studies using glial cell markers (Weiland et al., 1997Go), the present co-labeling data with NSE-IR provide the first positive demonstrations of the neuronal character of ER{alpha}- and ERß-bearing cells in the rat cerebral cortex. This labeling also revealed morphological clues as to whether receptor-bearing neurons were pyramidal or non-pyramidal. It was immediately evident, for example, that large and medium-sized cortical pyramids — the principal output cells of the cerebral cortex (Peters and Jones, 1984Go) — were immunonegative for both ER{alpha} and ERß. It was also evident that ERß-IR was sequestered within non-pyramidal neurons; in virtually every case, ERß-IR nuclei were found within small NSE-IR somata that radiated either bipolar or multipolar arrays of dendrites that bore striking resemblance to subsets of cortical neurons identified as GABAergic (Meinecke and Peters, 1987Go).

The characterization of ER{alpha}-IR neurons, however, was less clear. While ER{alpha}-IR cells residing in upper layers seemed to be non-pyramidal, those in deeper layers tended to display more ambiguous form. Because colocalization studies were limited to antigens preserved in the relatively weak fixative needed to optimize ERß-IR, double labeling with GABA, glutamic acid decarboxylase (GAD) and glutamate could not be used to clarify whether these cells were small excitatory pyramids and/or inhibitory local circuit neurons. Immunoreactivity for the calcium binding proteins PV and CB, which in rat cortex label subsets of GABAergic interneurons (DeFelipe, 1993Go), on the other hand, was well preserved. However, double-labeling studies revealed that almost without exception, ER{alpha}-IR cortical neurons were immunonegative for PV and for CB. In marked contrast, PV-IR identified an overwhelming majority of the ERß-IR cells in most sensorimotor and association areas, and together with CB seemed to account for most, if not all, of the ERß-IR cells in the allocortical piriform and entorhinal areas as well.

Localization of the classical estrogen receptor within GABA-ergic neurons has precedents in other brain structures. In the pre-optic area, for example, the majority of receptor-bearing neurons contain GAD, the synthetic enzyme for GABA (Herbison et al., 1993Go), while in the hippocampus, the laminar locations of ER-IR cells identify them as local circuit neurons (Weiland et al., 1997Go). Thus while neurochemical signatures of ER{alpha}-IR neurons in the cerebral cortex were not established in this study, there may be some anticipation that they correspond to subsets of GABAergic, local circuit neurons. Importantly, however, the negative findings obtained with respect to colocalization with CB and PV suggest that whatever their identity, ER{alpha}-IR neurons in the cerebral cortex are unlikely to overlap with ERß-IR cells. Although the possibility for a few individual cases of receptor isoform colocalization cannot be excluded, the present results suggest that at the cellular level, ER{alpha}- and ERß-mediated signaling streams are largely segregated in the adult rat cortex. Accordingly, few if any cortical neurons are likely to utilize estrogen's added signaling capacity afforded by the heterodimerization of ER{alpha} and ERß subunits (Pettersson et al., 1997Go; Ogawa et al., 1998Go). As discussed separately by receptor subtype below, the cellular segregation of ER{alpha} versus ERß signaling pathways appears to be further accentuated by regional and laminar differences in receptor distribution as well.

Regional and Laminar Localization of ER{alpha}-IR in Adult Rat Cortex: Comparison to Previous Studies and Functional Considerations

The very low levels of the classical ER may have discouraged focus on ER{alpha} as a subject for intensive, exclusive investigation in the mature cerebral cortex. Nonetheless, fragmentary data exist describing the distributions of ER{alpha} receptor proteins and mRNAs in the adult rat cortex. In most cases these data are consistent with details of the distribution of receptor-like immunoreactivity identified here. For example, the present results are a good match to a previous immunocytochemical study of the rat forebrain which included descriptions of small numbers of immunoreactive cells scattered among layers II/III of the cingulate and piriform cortices, and among layers II/III and IV/V of the lateral neocortex (Mufson et al., 1999Go). The present findings are also consonant with those from a second immunocytochemical study of rat hippocampus that additionally identified sparse complements of ER-IR cells particularly within the supragranular layers of retrosplenial and parietal cortex (Weiland et al., 1997Go). Curiously, however, in situ hybridization studies in adult rats consistently indicate that the few ER{alpha}-mRNA-containing cells identified are present mainly in layer IV (Simerly et al., 1990Go; Shughrue et al., 1997Go) — a layer in which neither ER{alpha}-IR nor ERß-IR cells were observed in this study.

While many studies have emphasized the paucity and scatter of cortical ER{alpha} cells, some novel features of ER{alpha}-IR identified here suggest precision in the distribution of this receptor isoform in the adult cerebrum. First, while perhaps scattered tangentially, in most areas ER{alpha}-IR cells took up highly stereotyped sublaminar locations, including the formation of a tier of receptor-positive cells that stood out along the superficial margins of layer II. Further, the insular cortices were unique sites of abrupt increases in the apparent density of ER{alpha}-IR cells, and dramatic shifts in their laminar distribution. Thus, while perhaps unimpressive for their number, the laminar locations and regional concentrations of ER{alpha}-bearing neurons suggests that they may nonetheless hold well-defined positions with respect to functionally specialized cortical subdivisions and circuits. The precise positioning of receptor-bearing neurons in the supragranular layers may further suggest some specific connection to the corticocortical communications that are mediated in part within these layers. Establishing the identity of ER{alpha}-IR cells, e.g. as projection or local circuit neurons, will be important next steps in drawing further inference about the cortical circuits and processes that may be direct targets of estrogen stimulation at the classical ER site.

Regional and Laminar Localization of ERß-IR in Adult Rat Cortex: Comparison with Previous Studies and Functional Considerations

The mRNAs encoding the ERß-receptor subtype, and, more recently, the receptor proteins themselves have been widely mapped in regions of the adult rat brain including the cerebral cortex (Shughrue et al., 1997Go; Shughrue and Merchenthaler, 2001Go). When compared with the present data, both descriptions reveal points of common ground as well as some disparities between the two types of analysis. Regionally, for example, immunocytochemistry and in situ hybridization are generally well aligned. Thus, in the isocortex, both ERß-mRNA and ERß-IR cells are localized mainly in deep cortical layers, and tend to be more abundant in lateral (sensorimotor) compared with medial (association) areas. Similarly, in the entorhinal cortex, receptor immunoreactivity and mRNAs are dense but more broadly distributed over major cellular layers (Shughrue et al., 1997Go). As for ER{alpha}, however, there are some discrepancies regarding the precise laminar distribution of immunoreactive and in situ hybridization signals for ERß. In all but the piriform and entorhinal cortices, for example, cells expressing ERß mRNAs are described as being present in layers IV–VI, while the ERß-IR cells identified here seemed to be more concentrated in (and in some cases, confined to) layer V, were virtually never seen in layer IV and were only rarely encountered in VI.

The recent findings of a relative abundance of ERß in the cerebral cortex has brought attention to this receptor subtype in particular as potentially explaining some of estrogen's effects in the adult cerebrum [see Kuiper et al. (Kuiper et al., 1998Go)]. The precise laminar and cellular localization of ERß-IR identified in this study add details that may be useful in further developing such functional hypotheses. For example, in the isocortex, ERß-IR neurons were concentrated in layer V, an infragranular stratum housing cortical outputs to subcortical motor centers such as the spinal cord, tectum, and neostriatum. Double-labeling studies indicated, however, that it is not the projection neurons themselves that are direct targets for receptor-mediated estrogen stimulation, but rather, subsets of the inhibitory interneurons that modulate their activity. Specifically, ERß-IR was present within interneurons that were largely immunoreactive for PV. This particular neurochemical signature in turn reveals the location of hormone signaling machinery as local circuit neurons that respond to depolarization with fast spiking, slowly adapting activity (Kawaguchi and Kubota, 1993Go) and which are presumably basket and chandelier cells [see DeFelipe (DeFelipe 1993Go)]; the latter are identifications that bring with them additional knowledge regarding stereotyped synaptic relationships and postulated roles in divisive and subtractive forms of inhibition (Blomfield, 1974Go; Somogyi et al., 1982Go). This information may combine with the regional differences in laminar distribution and areal densities of the PV cells observed in this study as receptor-bearing to produce specific predictions about the computations and functions that are directly influenced by estrogen activation at ERß sites in the adult isocortex. Although the specifics are far from clear, it may be predicted that very different arrays of cortical computations are influenced by ERß transactivation in the allocortex, since receptor immunoreactivity occupies different sets of layers, and colocalizes predominantly with CB-IR rather than PV-IR in these phylogenetically older areas.


    Summary and Conclusions
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Summary and Conclusions
 Notes
 References
 
The present study builds on previous hormone binding, immunocytochemical, and in situ hybridization studies to further understanding of the distribution of estrogen receptors in the adult cerebral cortex. As new details emerge, a common conclusion seems to be reinforced; that the {alpha} and ß isoforms of the intracellular estrogen receptor are differentially and precisely distributed among cortical regions, layers, and cells. Accordingly, each signaling stream seems to represent its own unique sphere of influence with respect to cortical structures and functions. Matching these advances in where estrogen stimulation may be brought to bear with more precise definitions of what this stimulation entails should bring important insights into observed estrogen effects on cortical information processing and perhaps on estrogen's postulated roles in instances of cortical dysfunction in adults [see Fink et al. (Fink et al., 1996Go)] as well. The current use of estrogen therapies in clinical settings and the development and application of estrogen receptor-specific pharmacology may make exploration of where and how estrogen signaling impacts the adult cerebrum timely as well.


    Notes
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Summary and Conclusions
 Notes
 References
 
The outstanding technical support of Mr Alex Adler is gratefully acknowledged. Mr James Tsiakos is also thanked for his assistance in initiating these studies. This work was supported by FIRST Award (R29NS35422) to MFK.


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 Introduction
 Methods
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 Discussion
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