Cerebral Cortex Advance Access originally published online on October 12, 2005
Cerebral Cortex 2006 16(8):1181-1192; doi:10.1093/cercor/bhj059
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Regional, Laminar and Cellular Distribution of Immunoreactivity for ERß in the Cerebral Cortex of Hormonally Intact, Postnatally Developing Male and Female Rats
Department of Neurobiology and Behavior, Stony Brook University, Stony Brook, NY 11794-5230, USA
Address correspondence to Mary Kritzer, Department of Neurobiology and Behavior, Stony Brook University, Stony Brook, NY 11794-5230, USA. Email: mkritzer{at}notes.cc.sunysb.edu.
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Abstract |
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Estrogen influences cerebral cortical development. Among the receptors involved are classical (ER
) and beta (ERß) intracellular estrogen receptors. In the first 2 weeks of postnatal life, cortical ER is transiently expressed at much higher levels than in adulthood. In this study, development of ERß was examined by mapping ERß immunoreactivity in relation to major cortical regions, layers and cell types in postnatal male and female rats that were 1–28 postnatal days (PND) old. These studies revealed that ERß-immunoreactive nuclei were present in the allocortices on PND 1 but were not detected in isocortex until PND 7. Allocortical labeling was also higher on PND 1 than at all later ages, while in isocortical areas low numbers of ERß nuclei were seen on PND 7 that rose to higher, near adult densities by PND 21. Finally, double labeling showed that ER was expressed mainly in neurons immunopositive for calretinin, while ERß was localized predominantly in parvalbumin-immunoreactive cells. Thus, the postnatal cortical developments of ERß and ER occur according to different timetables, different patterns and in association with different cortical cells. It thus seems it likely that the two also make distinct contributions to postnatal cortical development and/or sexual differentiation.
Key Words: allocortex • calretinin • ER • estrogen • isocortex • parvalbumin
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Introduction |
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Sex differences have been noted in the maturation of complex social, cognitive and mnemonic functions (Beatty, 1984
; Clark and Goldman-Rakic, 1989; Bachevalier and Hagger, 1991) in the incidence of developmental disorders such as schizophrenia where cortical operations are at risk (e.g. Seeman, 1996) and in the development of cortical structures including pyramidal cell dendrite branching, spine density and catecholamine innervation (Munoz-Cueto et al., 1990, 1991; Munoz-Cueto and Ruiz-Marco, 1994; Stewart and Kolb, 1994; Stewart and Rajabi, 1994). Taken together, these findings suggest that circulating gonadal hormones influence both the form and the function of the developing mammalian cerebral cortex. As in many reproductive brain centers (see Arnold and Gorski, 1984; Toran-Allerand, 1984), the sexual differentiation of the cerebral cortex is likely to be stimulated at least in part by sex-specific patterns of estrogen exposure. In rats this is influenced by the transient expression of aromatase, the enzyme responsible for the biosynthesis of estrogen from androgen, in the cerebral cortex during the first weeks of postnatal life (MacLusky et al., 1994). Because during this same time the testes are active in secreting testosterone and the ovaries are relatively inert, this creates an early one to two week postnatal period when estrogen, as the product of local androgen metabolism, is more abundant in the cerebral cortex of males. After roughly postnatal day 14 and prior to the onset of puberty, however, aromatase and testicular secretions decrease while the ovaries become increasingly active (Copechot et al., 1981; MacLusky et al., 1994) thus creating a later postnatal period when estrogen, as a secretory product of the ovaries, becomes more abundant in the cortex of females. These sex-specific patterns of estrogen availability are likely to exert important influences over the postnatal development and differentiation of the cerebral cortex. It is also probable that some of this influence is mediated via estrogen binding to its cognate intracellular receptors. The studies presented here describe the postnatal development of immunoreactivity for one of these — the beta subtype of the intracellular estrogen receptor (ER).
The cerebral cortices in both developing and mature rats contain the classical (ER) and the more recently identified beta subtype (ERß) of the intracellular estrogen receptor. These belong to a superfamily of ligand-activated transcription factors (Katzenellenbogen et al., 2000) that differ from one another structurally in their ligand binding and A/B domains (Kuiper et al., 1996), and functionally in their ligand selectivity (Kuiper et al., 1997), ability to induce distinct morphological and physiological endpoints (e.g. Patrone et al., 2000) and transcriptional activation of specific sets of genes (e.g. Paech et al., 1997). Mapping studies have also shown that in many regions of the central nervous system these receptor subtypes also differ in their distributions and relative abundance (e.g. Shughrue et al., 1997; Laflamme et al., 1998). This is the case in the adult cerebral cortex. In the rat cerebrum, ERß-immunoreactivity is fairly abundant and localized mainly to non-pyramidal neurons that are immunoreactive for the calcium binding protein parvalbumin (PV). In contrast, very few cortical neurons express immunoreactivity for ER, and those that do are PV-immunonegative (Blurton-Jones and Tuszynski, 2002; Kritzer, 2002).
Available evidence suggests that the cortical distributions and densities of ER and ERß also differ from one another during postnatal development. For example, in rats ER mRNAs and receptor proteins sharply rise during the first postnatal week, transiently peak at high levels, and then precipitously decline to extremely low levels similar to those seen in adults by about three weeks of age (e.g. Miranda and Toran-Allerand, 1992; Yokosuka et al., 1995; Guo et al., 2001; Hayashi et al., 2001; Perez et al., 2003). This contrasts the cortical development of ERß, where similar levels of ERß mRNAs are expressed in neonates and adults (Guo et al., 2001), and where ERß immunoreactivity appears later than that for ER and increases from initially low to progressively higher levels in older and adult animals (Zsarnovszky and Belcher, 2001; Perez et al., 2003). These maturational differences suggest that ER and ERß are likely to contribute in different ways and at different times to the development and sexual differentiation of the cerebral cortex, a possibility also supported by recent evidence suggesting that during postnatal development immunoreactivity for ER and for ERß may be localized in non-overlapping sets of cortical cells (Perez et al., 2003). The studies presented here sought more precise information as to where and when in the postnatal cortex the ERß subtype matures to help identify and parse roles for ER in cortical development.
Previous studies have shown that the ER that is transiently expressed in the cerebral cortex in early postnatal life is found in pyramidal and non-pyramidal neurons but not in glia cells (e.g. Yokusuka et al., 1995; Hayashi et al., 2001; Perez et al., 2003). It has been further shown that in the primary auditory cortex, many of the neurons that express ER are also immunoreactive for the calcium binding protein calretinin (CR, Hayashi et al., 2001). Less is known, however, about the cellular distribution of ERß in the developing cerebrum. To begin to provide this information, this study used methods of single label immunocytochemistry to define the distributions of ERß receptor proteins in male and female rats, 1–28 postnatal days (PND; day of birth designated PND 0) old, in relation to all major regions and layers of the cerebral cortex. Methods of double-label immunocytochemistry were then used to identify ERß immunoreactive cells as neurons and/or glia. Prompted by the high degree of colocalization observed in adult cortex (Blurton-Jones and Tuszynski, 2002; Kritzer, 2002), the anatomical relationships between ERß- and PV-immunoreactivity were also explored. Finally, the colocalization of ERß and ER was examined by first comparing single label immunocytochemistry for ER and ERß to identify cortical areas and postnatal ages where the two receptor subtypes were both present. Within these areas, double labeling was then used to determine whether ERß and/or ER immunoreactivity overlapped with that for PV and/or CR. Although these proteins identify non-overlapping subsets of neurons in the developing and adult rat cortex (see Hof et al., 1999), they were selected here for their potential as markers for ERß- versus ER-immunoreactive cells based the colocalization of ERß- but not ER- and PV-immunoreactivity seen in adult rat cortex (Blurton-Jones and Tuszynski, 2002; Kritzer, 2002), and on findings that as many as half of the neurons (pyramidal and non-pyramidal alike) expressing ER in the postnatally developing auditory cortex of rats are immunoreactive for CR (Hayashi et al., 2001).
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Materials and Methods |
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Tissue
A total of 39 male and 37 female Sprague–Dawley rat pups aged 1, 3, 5, 7, 10, 14, 21 or 28 PND were used. Each age group examined had a minimum of three male and three female subjects, and included individuals from at least two litters. All subjects were gonadally intact and housed with littermates; animals were housed with dams until PND 21. All procedures involving vertebrate animals were designed to minimize their discomfort and use, and are approved by the IACUC of Stony Brook University.
Tissue Preparation
Animals were deeply anesthetized with an i.m. injection of ketamine (150 mg/kg) and xylazine (50 mg/kg). After behavioral checks indicated an absence of deep reflexes, pups were transcardially perfused first with a 5–10 ml flush of phosphate buffered saline, pH 7.4, followed by phosphate buffered saline containing 2% paraformaldehyde and 15% saturated picric acid, delivered at low pressure through a small gauge needle for 10 min. Brains were then removed and post-fixed in the same fixative solution for 
6 h.
In view of previous findings of hormone receptor immunoreactivity suggesting greater antigen preservation in brain tissue immersion fixed in acrolein (e.g. Butler et al., 1999), four additional male and four additional female pups aged 5 and 7 PND were anesthetized as above, rapidly decapitated and their brains removed and immersion fixed for 10 h in 0.1 M phosphate buffered saline, pH 7.4, containing 5% acrolein. However, because no striking differences in cortical immunostaining for ERß, ER, GFAP, NSE and MAP-2 in immersion versus perfusion-fixed tissue were detected and because the quality of histology was far superior in the paraformaldehyde-fixed brains, immersion fixed brains were not used for the analyses or figures presented here.
Following fixation, brains were sunk in 0.1 M PB containing 30% sucrose for cryoprotection, were rapidly frozen in powdered dry ice, and stored at –80°C prior to sectioning on a freezing microtome. Tissue from animals aged 5 days or younger was cut at a thickness of 50 µm; that from all other subjects was cut at a thickness of 40 µm.
Single Label Immunocytochemistry
For each brain, separate series of sections were immunoreacted with antibodies for ERß (Zymed Laboratories, San Francisco, CA) or ER (Upstate Biotechnology, Lake Placid, NY). For these studies, sections were rinsed in 0.1 M phosphate buffer (PB), pH 7.4, reacted for 30–45 min in 0.1 M PB containing 1% H2O2, rinsed again in 0.1 M PB and then transferred to 50 mM Tris-buffered saline (TBS), pH 7.4. The sections were then transferred to a blocking solution composed of the TBS containing 10% normal swine serum (NSS) for 2 h at room temperature. After the blocking step, sections were incubated in the primary antibody (diluted 1:1000 for ERß and 1:6000 for ER in 50mM TBS containing 1% NSS) for 3 days at 4°C. Following this incubation, sections were rinsed carefully in TBS, placed in biotinylated secondary antibodies diluted 1:100 in TBS containing 1% NSS for 2 h at room temperature, were rinsed again in TBS and were then placed in avidin-biotin complexes horseradish peroxidase solution (ABC, Vector Labs, Burlingame, CA) for an additional 2 h at room temperature. Afterward, sections were rinsed in TBS and in Tris buffer (TB), pH 8.0, and were reacted using 0.07% 3.3'-diaminobenzidine (DAB) and 0.006% nickel ammonium sulfate as chromagen.
Double Label Immunocytochemistry
Sections were also reacted sequentially, first using one of the anti-ER polyclonal antibodies (above), and then using one of several monoclonal antibodies recognizing cell subtype-specific antigens. The procedure used for labeling ER was as above but was followed by rinses in 50 mM TBS and then an incubation step in blocking solution (TBS containing 10% NSS; either 2 h at room temperature or overnight at 4°C). Following this, the sections were placed in a monoclonal antibody diluted in TBS containing 1% NSS (1 day, 4°C). The monoclonals and their working dilutions were as follows: anti-neuron specific enolase (NSE, Chemicon International, Inc., Temecula, CA, 1:100), microtubule associated protein-2 (MAP-2, Sigma Chemical Co., St. Louis, MO, 1:500), glial fibrillary acidic protein (GFAP, Chemical International Inc., 1:300), parvalbumin (PV, Sigma, 1:1000) and calretinin (CR, Sigma, 1:1000). After incubation in primary antibody, sections were rinsed and reacted using DAB alone (0.07%) as chromagen. All immunoreacted sections (single- and double-labeled) were then slide mounted, dehydrated and placed under coverslips; representative sections were also lightly counterstained for nissl substance using 1% cresyl violet to reveal cytoarchitecture.
Control Experiments
The immunocytochemical procedures described above were carried out on representative sections with the omission of primary or secondary antibodies (e.g. Fig. 1I). For ERß immunostaining, some control sections were also reacted using antiserum that had been preabsorbed with 10 µM of the immunizing peptide (see Creutz and Kritzer, 2004).
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Data Analysis
Qualitative assessments of labeling were made in each animal subject in representative rostrocaudal series of sections encompassing all major fields of the cerebral cortex. These qualitative studies included evaluation of the appearance and intensity of ER- and ERß-immunostained cells, their regional and laminar distributions and their approximate regional densities. In animals aged 5 days and older, colocalization of receptor immunoreactivity with that for a series of cell subtype-specific cytoplasmic markers (MAP-2, NSE, GFAP, PV, CR) was also examined. In all cases, cortical areas and layers were cytoarchitectonically defined as per Zilles and Wree (1985).
Quantitative estimates of the data were also obtained. These consisted of counts of all visible ERß immunoreactive nuclei (single and/or doubly labeled with PV) that were present per unit area, measured in at least two male and two female subjects in representative cortical regions in animals aged 1, 7, 14 and 21 postnatal days of age. The regions analyzed were the piriform and entorhinal areas of the medial temporal lobe, and areas Cg1, Fr1, Par 1 and OC1B of the lateral and dorsomedial cortex. Immunoreactive nuclei were counted using a Zeiss Axioskop (Carl Zeiss, Inc., Thornwood, NY) under brightfield illumination using a x20 objective and a calibrated eyepiece reticle (150 x 300 µm) to delineate a counting rectangle. In piriform cortex, this counting frame was placed over layer III, in entorhinal cortex over layers III–V and in each of the isocortical areas evaluated, over layer V; in all regions, the counting box was oriented with its long edge parallel to cortical layering, and counts were made of all immunoreactive nuclei appearing within the box throughout the full thickness of the tissue section and using its top and right sides as exclusion lines. This counting procedure was repeated five times across at least four separate tissue sections per animal per area, with sections matched for anterior/posterior level across subjects using internal and external structures as landmarks. No other attempts were made to pre-select counting regions within a given area. Counts were not corrected for tissue shrinkage, cell size or overall cell density and did not adhere to formally defined principles of random sampling. Accordingly, values were subjected to descriptive statistical analyses (mean, variance, standard deviation, standard error) only (Stat View 4.5). These methods replicate those used in conjunction with a previous, mainly qualitative study to estimate the densities and degrees of double labeling of ERß-immunoreactive (-IR) nuclei in corresponding areas of the adult rat cerebral cortex in which three male subjects, two proestrus females and three diestrus females were analyzed (Kritzer, 2002); most of these quantitative data are previously unpublished and appear here for purposes of comparison for the first time. All counts were performed on slides that had been coded prior to the analysis and all assessments were made by a single observer (M.F.K.), who was blind to the adult animals' sex and hormone status, and blind to the age and sex of developing animals.
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Results |
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This study charted the postnatal development of ERß in relation to all major regions, layers and cell types of the cerebral cortex in postnatally developing rats. Eight postnatal age groups from PND 1 to PND 28 were evaluated. For each age group examined, separate evaluations were made of left and right hemispheres of identified male and female subjects. However, no obvious hemispheric or sex differences were noted. Further, counts of ERß-IR nuclei made per unit area in corresponding, representative cortical regions of PND 1, 7, 14 and 21 animals revealed values in males and females that were almost all within 10% of one another (see Tables 1 and 2). Accordingly, the descriptions below should be assumed to apply to both cerebral hemispheres and to both male and female subjects.
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Regional and Laminar Distribution of ERß
ERß-immunopositive cells were identified in the cerebral cortex of postnatally developing rats at the earliest age examined, PND 1. Throughout the entire postnatal period evaluated, immunopositive cells were characterized by immunoreactivity that was concentrated over the cell nucleus with little to no above background labeling in the surrounding cytoplasm (see Fig. 1).
During the first postnatal week, ERß-immunoreactivity was present only in piriform and entorhinal cortex, i.e. the allocortices of the medial temporal lobe (Fig. 2). Within these regions, the immunoreactive nuclei observed were typically round in shape and between 5 and 8 µm in diameter. On PND 1 their staining intensity varied from faint to extremely dense. By PND 3, however, the majority of the ERß-immunoreactive (IR) nuclei present were strongly immunoreactive (Fig. 1A–D).
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From PND 1 to PND 3, the immunoreactive nuclei present in both allocortical regions evaluated were numerous; ERß-IR nuclei were dense but scattered without discernible anteroposterior or mediolateral gradients in layer III of piriform cortex, and in layers II–V of entorhinal cortex (Fig. 2). By PND 5, however, a decrease in the density of these nuclei was apparent in both allocortical fields, and over the next days to weeks, the apparent densities of labeling in both piriform and entorhinal regions continued to decline to levels reminiscent of the moderately diffuse ERß-immunostaining present in corresponding areas of the adult rat allocortex (e.g. Fig. 3A–C; Kritzer, 2002
). Density measures of ERß-immunoreactive nuclei bore this out, revealing between 40–60 immunoreactive nuclei per unit area on PND 1, and densities that dropped by >50% by PND 7 and by nearly half again by PND 14 to reach and remain at values of between 7–10 nuclei per unit area. These latter values were comparable to those measured in corresponding areas of mature male and female animals (Table 1).
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ERß-IR nuclei were not observed outside of the allocortices until PND 7. While just 2 days earlier (PND 5) no immunoreactive nuclei were observed in any cortical areas beyond the medial temporal lobes, on PND 7 small numbers of faintly labeled profiles appeared virtually simultaneously across all of the major sensory, motor and association areas of the lateral and dorsomedial cortex (e.g. Fig. 4). These more recently appearing nuclei were mostly round and had diameters similar to or slightly smaller than those present in entorhinal or piriform cortices (4–7 µm across). However, also scattered among these were subpopulations of ERß-IR nuclei that had diameters that were noticeably larger (10–15 µm across; Fig. 1E–H).
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In each of the sensory, motor and association areas examined, the initial (PND 7) laminar location of ERß-IR nuclei was predominantly layer V (Fig. 4, first column and Fig. 5A–C). Over the next week (PND 7–14) the number and the staining intensity of the ERß-IR nuclei in this infragranular layer increased, and in sensory and motor areas small numbers of immunoreactive nuclei also began to appear in layers I, II/III and VI (Fig. 4, second column and Fig. 5D–F). These trends continued such that by the end of the third postnatal week (by PND 21) the patterns of immunoreactivity in all major regions of the developing cerebrum resembled those observed in corresponding regions of the adult (Fig. 4, third column and Fig. 5G–I). Qualitatively, for example, fairly low numbers of strongly ERß-IR nuclei were present in layer V and to a lesser extent layer in VI in the medial prefrontal, cingulate and insular cortices; in motor and premotor areas nuclei were most dense in layer V and scattered more diffusely in layers I, II/III and VI; and in primary and secondary sensory cortices, e.g. areas Oc1B and 2L, Par 1 and 2, Te1 and 2, ERß-IR nuclei were most dense in the lower half of layer V and more sparsely distributed in layers I, II/III and VI (see Fig. 4). Quantitative assessments of the density of immunoreactive nuclei in layer V of representative regions confirmed the progressive postnatal increase in ERß-immunoreactivity from low density values measured on PND 7 to steadily higher values measured over the next one to two weeks that approached those measured in corresponding areas of adult cortex (Table 2).
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Cellular Distribution of ERß
The cells that were immunoreactive for ERß appeared to be neurons. First, at each postnatal age studied the ERß-IR nuclei had diameters (4–12 µm; see Fig. 1) that were more than twice what is typical for most cortical glial cells (see Fig. 6E,F). Further, double-label immunocytochemical studies, limited to PND 5 animals — when immunoreactivity for cytoplasmic markers first became detectable — revealed colocalization between ERß and the neuronal markers MAP-2 (Fig. 6A,B) and NSE but not between ERß and the glial cell marker GFAP (Fig. 6E,F).
In both allo- and isocortex, most of the NSE- or MAP-2-IR neurons that were immunoreactive for ERß showed non-pyramidal morphology. In the adult cortex ERß-immunoreactivity colocalizes predominantly with subsets of PV immunoreactive non-pyramidal neurons (Blurton-Jones and Tuszynski, 2002; Kritzer, 2002). To investigate this relationship in the developing cerebrum, double-labeling studies using anti-ERß and anti-PV antibodies were also carried out. Similar to what has been previously reported, PV-immunoreactivity appeared in the postnatal cerebrum in different regions on different days (e.g. Alcantara et al., 1993), and in most areas clear, cellular immunoreactivity for PV appeared just days after that for ERß. However, as soon as distinct PV-IR neurons could be detected, colocalization with ERß-immunoreactivity was observed. Beginning on PND 7, for example, when mature-looking PV-IR neurons were first apparent in the allocortex, counts of ERß-IR and ERß/PV-IR cells made per unit area revealed that between 1 in 5 and 1 in 10 of the ERß-positive nuclei present in a given field were surrounded by PV-IR cytoplasm. Similar degrees of ERß/PV colocalization were also observed at later time periods, despite substantial decreases in the densities of ERß-immunoreactive nuclei (Table 1). The values measured at all representative postnatal ages were similar to those previously reported for the allocortices in adult rats (Kritzer, 2002).
In the more lateral and dorsomedial regions of the cortex, where densities of ERß-IR nuclei increased with age (Table 2), so too did the proportions of receptor-immunopositive nuclei that colocalized with PV-immunoreactivity. For example, PV-IR cells were identified earliest in rostral area Par 1 on PND 7; in this region quantitative analyses revealed that only about half of the ERß-IR nuclei present within a given field were also PV-immunoreactive (e.g. Fig. 7G). However, by the end of the third postnatal week (PND 21), the proportions of ERß-IR cells present within corresponding cortical areas had risen to between 80 and 90%. Similar progressions of ERß/PV colocalization from initial values of 50% to between 80 and 100% were also documented for areas Cg1, Fr1 and OC1B, although in these areas the progression followed timetables that were delayed by the later appearance of PV-immunoreactivity (e.g. Fig. 7). In all cases, however, ERß- and PV-colocalization plateaued at values of between 80 and 100% by PND 21; these values are comparable to those previously reported for corresponding cortical areas of the cerebral cortex of adult rats that were derived using the same methods of quantitative assessment (Kritzer, 2002).
ER-immunoreactivity
Previous studies have described a transient surge in the expression of ER-IR in the cerebral cortices of postnatally developing rats (e.g. Yokosuka et al., 1995; Hayashi et al., 2001). Consistent with these studies, ER-immunoreactive nuclei first appeared in the subjects of this study in large numbers and across widespread areas of the cortex between PND 3 and 5, peaked in density between PND 7 and 10, and then declined to very low levels of labeling characteristic of the adult cerebrum by three weeks of age. The question asked here was whether ER-immunoreactivity colocalized with that for ERß. Because the anti-ER and ERß antibodies used in this study were both polyclonal antibodies generated in rabbits, an indirect approach of comparing the phenotypes of ER versus ERß-bearing cells was adopted. Specifically a series of double-labeling studies were carried out in representative cortical areas in rats between 5 and 14 days of age. Initially, anti-ER antibodies were used in combination with neuronal and glial cell markers. These analyses confirmed previous findings that ER-immunoreactivity was expressed in both pyramidal and non-pyramidal neurons but not in glial cells. To determine whether the non-pyramidal ER-IR neurons might be those immunopositive for ERß, colocalization between ER- and PV-immunoreactivity was next examined. However, in contrast to the >50% overlap seen between ERß- and PV-IR (e.g. Fig. 6J), there was no evidence for cellular colocalization between anti-ER and anti-PV antibodies (Fig. 6K). Finally, in view of recent evidence for a roughly 50% degree of colocalization between ER-IR and immunoreactivity for the calcium-binding protein calretinin (CR) in developing rat cortex (Hayashi et al., 2001), antibodies recognizing ER or ERß and CR were also combined. These analyses revealed substantial overlap between CR- and ER-immunoreactivity in the developing cortex (Fig. 6L) but virtually no colocalization of CR- and ERß-immunolabeling (Fig. 6M).
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Discussion |
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Numerous endpoints in the developing cerebral cortex have been identified as estrogen-sensitive. In rats in vitro studies have shown that estrogen stimulates neuronal differentiation, neurite extension and cell survival in organotypic and dissociated cell cultures (Toran-Allerand, 1976
, 1984; Brinton et al., 1997; Zhang et al., 2000) while hormone manipulations have revealed estrogen influences on the development of pyramidal cell dendrite arborization and spine density, and on cortical catecholamine innervation in vivo (Munoz-Cueto et al., 1990; Stewart and Kolb, 1994; Stewart and Rajabi, 1994). Previous studies examining development of the estrogen receptors that are likely to be involved in these processes have focused mainly on the classical intracellular estrogen receptor, ER. In the present study, methods of single- and double-label immunocytochemistry were used to obtain detailed information about the cortical maturation of the more recently identified ERß. In the sections that follow, details of the distributions, densities and dynamics of cortical ERß development that were found are compared to findings from previous developmental studies of ERß in the rat cortex, are contrasted with those for ER, and are considered from functional and finally from cellular perspectives. These sections are preceded, however, by a more general discussion of the sex differences, or more specifically the lack thereof, noted in the postnatal development of cortical ER. Development of ERß-IR in the Cerebral Cortex of Male and Female Rats
The permanent sexual differentiation of the structure and function of reproductive, neuroendocrine as well as higher order brain areas in rats and other mammals is stimulated in large part by their differential exposure to testosterone in developing males and females (see Arnold and Gorski, 1984; Toran-Allerand, 1984). However, it is often testosterone aromatized to estradiol and the interaction of this metabolite with intracellular estrogen receptors that is responsible for the differentiating hormone actions observed (see MacLusky and Naftolin, 1981; Arnold and Gorski, 1984; Toran-Allerand, 1984). In many areas these sex differences in estrogen exposure are also accompanied by sex differences in the abundance of intracellular estrogen receptors. In rats, for example, analyses in hypothalamic, limbic and other subcortical brain areas have revealed striking sex differences in estrogen binding, receptor immunoreactivity and/or receptor mRNAs for both ER and ERß in developing animals that in some cases persist through adulthood (e.g. Brown et al., 1988; Kuhnemann et al., 1994; Orikasa et al., 2002; Ikeda et al., 2003). It thus seems likely that within these subcortical regions, sex differences in estrogen availability and estrogen receptors are both factors that can act synergistically or otherwise in imposing sex differences on the developing brain and stimulating sex-specific behaviors later in life. In the cerebral cortex, however, studies in rats using immunocytochemistry (Yokosuka et al., 1995; Kritzer, 2002; Perez et al., 2003) or in situ hybridization (e.g. Shughrue et al., 1990; Guo et al., 2001) to quantify ER and ERß have revealed no discernible sex differences in the developmental or adult expressions of these receptor markers (but see Sandhu et al., 1986). In this study as well, separate evaluations of ERß-immunoreactivity in male and female subjects revealed no apparent sex differences in the laterality, tempo or other qualitative or quantitative aspects of postnatal cortical ERß development. Thus, in contrast to brain areas where coincidental sex differences in estrogen availability and in estrogen receptors may both act to either potentiate or limit estrogen signaling, in the cerebral cortex sexual differentiation may be more singularly dependent on sex-specific patterns of estrogen availability. This could render the developing cortex especially vulnerable to anomalies in hormone secretion and/or metabolism. Such vulnerability could provide a basis for the sex differences in the cortical symptoms that are observed in certain developmental disorders, e.g. schizophrenia, and may also offer insights as to why these sex-specific cortical deficits often occur in the absence of obvious defects in reproductive or neuroendocrine axes (see Seeman, 1996).
ERß in the Postnatal Rat Cerebral Cortex: Comparison to Previous Studies
Among the main findings of this study was the identification of very different postnatal courses of development for ERß-immunoreactivity in entorhinal and piriform compared to other areas of the cerebral cortex. Specifically, in the two allocortical areas examined, ERß-IR nuclei were already present on PND 1 (the earliest time point evaluated) and were observed initially at higher densities than the more moderate levels of labeling that characterize these areas later in postnatal and adult life (Table 1). In contrast, in the more lateral and dorsomedial cortex, ERß-immunoreactivity was not detected until PND 7, and in all regions progressed from initially low densities to higher concentrations of immunoreactive cells that leveled off by two to three postnatal weeks of age at or near the moderate densities seen in corresponding areas of adults (Table 2). Both sets of observations have precedent in the two previous studies that also examined ERß-immunoreactivity in the postnatally developing cerebral cortex of rats. In the first, ERß receptor proteins were examined primarily in the barrel field representation of the primary somatosensory (Par1) cortex of female animals. This study described ERß-immunoreactivity in this isocortical field as absent on PND 3, sparse on PND 6 and rising to substantially higher levels at older ages (PND 18, 25, adult), and also noted that moderate levels of ERß immunoreactivity were present in the perirhinal and piriform cortices on PND 3 (Zsarnovszky and Belcher, 2001). In the second study, ERß-immunoreactivity was examined more cortically wide and in males and females, but only on PND 3 and PND 14; this study reported ERß-IR nuclei in the allo- but not isocortex on PND 3, and in the allocortex as well as infragranular layers (V and VI) of all major isocortical fields on PND 14 (Perez et al., 2003).
Thus, there is good agreement across studies that within the postnatal cortical mantle, ERß proteins appear earlier in allocortical compared to other cortical fields. Other differences distinguishing ER- and/or ERß-immunostaining across these two classes of cortex were also identified in this study. For example, double-labeling studies carried out here and in a previous study of the adult cortex (Kritzer, 2002) suggest that the cells that are immunopositive for ERß in the allocortices are phenotypically distinct from those present in the isocortex. Specifically, whereas 80–100% of the ERß-IR cells in the lateral and dorsomedial neocortex are PV-IR (Blurton-Jones and Tuszynski, 2002; Kritzer, 2002; present study), in entorhinal and piriform areas less than half are PV-IR at any postnatal stage (Kritzer, 2002). Further, ERß-IR cells in the allo- versus isocortices showed contrasting patterns of densities with increasing age, whereas in isocortical fields, ERß-immunoreactivity became more and more abundant as postnatal maturation proceeded.
There is less concordance among studies regarding the distributions and densities of ERß-IR cells, particularly within isocortical fields. For example, in this study, semi-quantitative analyses of ERß-IR nuclei in area Par1 revealed that immunoreactive cells were most numerous in layer V, and peaked in density in this layer at adult-like, moderate values of roughly 15 immunoreactive nuclei per 450 µm2 area by about PND 21. In contrast, the previous study in barrel field cortex which used similar methods to those used in this study to estimate immunoreactive cell density identified much higher concentrations of ERß-immunoreactive cells. Specifically, ERß-immunolabeling increased from scattered cells on PND 6 to between 36 and 60% of all cortical neurons present per unit area by PND 18, between 29 and 55% of all neurons by PND 25, and between 30 and 60% of all neurons in adult animals (Zsarnovszky and Belcher, 2001). Although these representations of cell density utilize different terms, comparisons of photomicrographs alone indicate that the quantitative differences in the amounts of cortical immunostaining between this and the present study are considerable. Qualitative differences in the laminar and cellular distributions of immunolabeling also separate these studies. For example, while the present study and those describing ERß-immunoreactivity in adult cerebrum found receptor immunoreactivity predominantly in the nuclei of non-pyramidal neurons in the infragranular cortical layers (Blurton-Jones and Tuszynski, 2002; Kritzer, 2002), in the developing and mature barrel field ERß-immunoreactivity was observed in pyramidal as well as non-pyramidal neurons, was abundant in all major cellular layers, and included populations of cells in which immunoreactivity was concentrated over the cytoplasm as well as the nucleus (Zsarnovszky and Belcher, 2001).
In considering the origins of these study-to-study disparities, it seems unlikely that the difference noted were a consequence of investigating different portions of the body map in area Par1, as qualitative assessments in this study included the whole of area Par1 and revealed no cortical areas where ERß immunoreactivity was as abundant or widespread as that previously described for the barrel field (Zsarnovszky and Belcher, 2001). Differences in methods of fixation also seem unlikely to have produced these differences; although the previous study used immersion fixation in a paraformaldehyde/acrolein solution, we found no apparent quantitative or qualitative differences among age-matched, immunoreacted tissue sections obtained from animals that were transcardially perfused with paraformaldehyde versus immersion fixed in acrolein (see Materials and Methods). However, the use of different antibodies to label receptor proteins should be taken into account, particularly as findings from two developmental studies (Perez et al., 2003; present study) and two studies in adult rat cortex (Blurton-Jones and Tuszynski, 2002; Kritzer, 2002) in which a common commercial antibody (Zymed) was used all report qualitatively and quantitatively similar results that also closely match cortical distributions reported for ERß mRNAs (e.g. Shughrue et al., 1997).
ERß in the Developing Cerebral Cortex: Comparison to ER and Functional Considerations
Since the discovery of the second major subtype of the intracellular estrogen receptor (Kuiper et al., 1996), comparative studies have identified differences in sequence structure, ligand binding, transcriptional activation properties (see Katzenellenbogen et al., 2000) and CNS distributions (e.g. Shughrue et al., 1997; Laflamme et al., 1998) that together suggest that ER and ERß play very different roles in stimulating brain form and function. This seems likely in the case of the cerebral cortex, where not only do these two signaling streams occupy anatomically and functionally distinct cellular niches in adults (Blurton-Jones and Tuszynski, 2002; Kritzer, 2002), but, as discussed below, in much of the cortical mantle they mature postnatally in different ways, according to different timetables, and in association with non-overlapping sets of cells.
The most striking contrast in the postnatal cortical development of ER versus ERß lies in the relative abundance of these two receptor subtypes. Specifically, while in the adult cerebrum ERß is more prevalent than ER, the opposite is true perinatally. Thus, in mice, rats and primates, striking surges in binding, mRNAs and immunoreactivity for ER are seen during the first weeks of postnatal life (e.g. MacLusky et al., 1979; Sheridan, 1979; Gerlach et al., 1983; Sandhu et al., 1986; Shughrue et al., 1990; Miranda and Toran-Allerand, 1992; Yokosuka et al., 1995; Guo et al., 2001; Hayashi et al., 2001; Perez et al., 2003), and in rats, these ER markers peak at levels comparable to those seen in hypothalamic structures between PND 7 and 10. In contrast, during this same timeframe, in most areas of the cortex immunoreactivity for ERß is only beginning to be detected and is present at very low levels (Zsarnovszky and Belcher, 2001; Perez et al., 2003). Further, in contrast to the sharp perinatal rise and precipitous decline in ER, the semi-quantitative analyses presented here and previously (Zsarnovszky and Belcher, 2001) indicate that the densities of ERß-IR nuclei increase steadily from low to higher densities to reach near adult levels by between 21 and 28 postnatal days of age.
Because the postnatal cortical peaks in ER and ERß occur at different times they are also coincident with different sets of developmental events. For example, peaks in ER occur around PND 7, which is roughly the same time that maxima in processes of cortical cell migration, differentiation, myelination and synaptogenesis are observed (see Uylings et al., 1990). Further, that cortical ER also shows a systematic developmental shift in receptor localization from deeper to more superficial cortical layers during this same postnatal time frame (e.g. Shughrue et al., 1990; Miranda and Toran-Allerand, 1992; Yokosuka et al., 1995) strengthens the relationship between ER and several of these processes, e.g. radial cell migration and neuronal differentiation, which also follow infragranular to supragranular maturational gradients (see Uylings et al., 1990). Finally, because ER also peaks in parallel with cortical aromatase activity (MacLusky et al., 1994), it is not difficult to envision how the selective stimulation of this receptor subtype could also yield sex differences such as the increased neuron numbers found in visual cortices of male compared to female rats (Reid and Juraska, 1995) and the transiently greater numbers of axodendritic synapses in the cerebral cortices of postnatally developing female compared to male rats (Munoz-Cueto et al., 1991; Munoz-Cueto and Ruiz-Marco, 1994) despite the presence of similar levels of receptors in male and female subjects.
Immunoreactivity for ERß, on the other hand, emerges and peaks in density later in postnatal life, during a period when more subtle refinements in cell structure and connectivity are taking place. The localization of ERß mainly to the infragranular layers and to PV-IR cells further suggests that its postnatal influence may be selective for particular cortical layers and cell types. Evidence from ERß knockout mice and from organotypic cultures of postnatal rat cortex provide some support for this scenario. For example, ERß knockout mice examined on PND 14, PND 30 and at 1 year of age show marked cortical hypocellularity, migrational failure of neurons destined for the supragranular cortical layers, and morphological abnormalities in radial glial cells and astrocytes (Wang et al., 2001, 2003). These findings suggest that ERß-mediated influences are important for processes of radial cell migration and apoptosis. However, because the cortical layers and cell types that are most affected contain little to no ERß-immunoreactivity in postnatal life, the relevant stimulation may more likely occur prenatally or involve brain areas that are afferent to the cortex. In contrast, however, at 2 years of age, the primary cortical defect observed in ERß knockout mice is cortical cell loss that is most prominent in layers IV and in layer V (Wang et al., 2001). The latter layer, layer V, is where immunoreactivity for ERß is most concentrated in postnatally developing and adult rat cortex. Thus it may be that the later cortical effects observed in ERß knockout mice are related to a loss of estrogen stimulation at the infragranular ERß sites that are normally present in the postnatal cortex.
The infragranular concentration of ERß-immunoreactivity in the postnatal rat cortex thus provides a good fit with the laminar patterns of hypocellularity that are induced posnatally in ERß knockout mice (Wang et al., 2001). Studies in organotypic cultures from postnatal rat cortex, on the other hand, have identified effects of estrogen stimulation that seem to map even more specifically onto the cells that are immunoreactive for ERß. Specifically, the addition of estrogen to cultures derived from frontal cortex of PND 2 and PND 3 rats significantly stimulates the dendritic length, dendritic branching, and migration and/or expression of PV-IR cells in supra- and infragranular cortical layers (Ross and Porter, 2002). Because in vivo PV-IR cells in the postnatal cerebrum can be immunoreactive for ERß but are immunonegative for ER, it seems likely that estrogen's effects on the maturation of cultured PV-IR cortical neurons are mediated by ERß. Further exploration of this issue is warranted not only for its potential to identify an example of an ERß-mediated, ER independent action of estrogen in the developing cerebral cortex, but also for its possible relevance for schizophrenia where sex-specific patterns of cortical deficits have led to hypothesized roles for estrogens (e.g. Seeman, 1996) and where PV-IR interneurons neurons in certain parts of the cortex appear to be especially at risk (Lewis et al., 2005).
Cellular Localization of ERß and ER in Postnatally Developing Cerebral Cortex
Although their peaks are well separated in time, there are spans during postnatal cortical development when ER and ERß are both relatively abundant in the cerebral cortex. However, even during these limited periods, a division of labor between ER and ERß in mediating processes of cortical maturation may still be anticipated based on their expressions in what appear to be non-overlapping populations of neurons. Thus, while, in the adult, ER is found in small numbers of PV-immunonegative, non-pyramidal neurons, during its peak in early postnatal life this receptor is transiently expressed in large numbers of pyramidal and non-pyramidal neurons, many of which are CR-IR but not PV-IR (Hayashi et al., 2001; present study). In contrast, during nearly all stages of postnatal life, ERß-immunoreactivity is expressed predominantly in subsets of PV-IR non-pyramidal neurons, while in the developing animals examined in this study immunoreactivity for ERß was never seen in CR-IR cells.
In addition to differential anatomical localization (above), there are also intriguing parallels in the developmental progressions of ER and CR-immunoreactive neurons on the one hand and ERß-immunoreactive cells and PV-immunoreactive neurons on the other. Specifically, like ER-IR cells, CR-IR neurons peak in density in the postnatal rat cerebrum at about PND 7, transiently include large numbers of pyramidal and non-pyramidal cells, and sharply decrease from peak densities in early postnatal life to much lower, near-adult values and exclusive non-pyramidal cell localizations by PND 15–21 (Fonseca et al., 1995; Schierle et al., 1997; see Hof et al., 1999). In contrast, like ERß-IR cells, the invariably non-pyramidal parvalbumin-IR neurons of the developing rat cerebrum are identified initially in low numbers in deeper cortical layers later in postnatal life (around PND 7), and from there steadily increase in number, density and laminar distribution to reach mature patterns of labeling by the end of the third postnatal week (Solbach and Celio, 1991; Sanchez et al., 1992; Alcantara et al., 1993, see Hof et al., 1999).
Findings of ERß but not ER within PV-IR neurons and of ER but not ERß within CR cells support conclusions from a previous double-labeling study that found no evidence for cellular coincidence between immunoreactivity for ER and ERß in any major region of the cortex in PND 3 or PND 14 male or female rats (Perez et al., 2003). These observations combine with similar findings obtained in adult rats (Kritzer, 2002) to suggest that it is highly unlikely that the cerebral cortex makes significant use of the additional signaling capacity afforded by ER/ERß heterodimerization (Pettersson et al., 1997; Ogawa et al., 1998) at any time during postnatal life. The cellular separations of ERß and ER also add to other contrasts such as the earlier detection and transient peaks in ER versus later appearance and progressive increase in ERß, to predict largely if not entirely distinct contributions of these two intracellular receptor subtypes in mediating estrogen's effects on the postnatal development and sexual differentiation of the cerebral cortex. The ERß-mediated stimulation of PV-IR cells discussed above may thus be only one of many endpoints that are modulated by ER subtype-specific stimulation in the postnatally developing cerebral cortex.
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This work was supported by NS41966 to M.F.K., and in collaboration with the Cody Center and Stony Brook Initiative for Autism Research. The outstanding technical support of Ms Aiying Liu is also gratefully acknowledged.
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