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ARTICLE |
and N-Methyl-D-Aspartate Receptor in the Hypothalamus of Female Rats
Kastor Neurobiology of Aging Labs, Fishberg Research Center for Neurobiology and Brookdale Department of Geriatrics and Adult Development, Mount Sinai School of Medicine, New York, New York 10029
Address all correspondence and requests for reprints to: Andrea C. Gore, Ph.D., Neurobiology of Aging Laboratories, Mount Sinai School of Medicine, Box 1639, New York, New York 10029-6574. E-mail: andrea.gore{at}mssm.edu.
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Abstract |
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, in the anteroventral periventricular nucleus and the medial preoptic nucleus, two critical regions for reproductive physiology and behavior. We observed extensive colocalization of ER and NR1 in these brain regions (
80%). In the anteroventral periventricular nucleus, treatment of ovariectomized rats with estrogen up-regulated the coexpression, whereas in the medial preoptic nucleus, estrogen had no effect, demonstrating a regional specificity to the estrogen sensitivity. The number of ER cells that did not express NR1 was not altered by estrogen treatment in either brain region. Thus, we speculate that the extensive colocalization of ER and the NMDAR provides an anatomical level at which estrogen and glutamate can act at target cells, and potentially synergize, to influence neuroendocrine and autonomic functions. |
Introduction |
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that is abundant in the hypothalamus but absent in GnRH neurons (4, 5, 6). Among the numerous neurotransmitters regulating GnRH neurons is glutamate, acting through the N-methyl-D-aspartate (NMDA) receptor (NMDAR). Activation of NMDARs stimulates GnRH release and gene expression at puberty (7, 8, 9, 10, 11) and is involved in the preovulatory GnRH/LH surge (12). As is the case for estrogen, actions of glutamate through the NMDAR on GnRH neurons are likely to occur both directly and indirectly. Recent studies indicate that GnRH neurons express NMDARs, and this is regulated by developmental stage (9, 13, 14, 15). In addition, the hypothalamus and preoptic area is abundant in NMDARs on other, non-GnRH cells, some of which could make projections to GnRH perikarya or neuroterminals (9, 13).
The effects of NMDAR activation on GnRH neurons are dependent upon the steroid hormone environment. NMDAR agonists stimulate GnRH or LH release in estrogen treated, ovariectomized (OVX) rats (16, 17); these effects are significantly reduced in OVX rats in the absence of estrogen replacement. In addition, treatment with an NMDAR antagonist blocks the steroid-induced GnRH/LH surge (11, 12), indicating a potential interaction between estrogen and glutamate. However, NMDAR binding is not influenced by estrogen in the hypothalamus (18), suggesting that glutamate and estrogen may interact by some other mechanism to affect GnRH neurons and reproductive function (19). Therefore, we speculated that there might be an anatomical site at which these factors interact to result in this facilitation of NMDAR activation by estrogen.
In this study, we examined the expression of ER and its colocalization with the NMDAR in two regions critically involved in reproductive function: the anteroventral periventricular nucleus (AVPV) and medial preoptic nucleus (MPN). These regions were chosen because they are critical for normal reproductive physiology and behavior (20, 21, 22). The AVPV and MPN are a source of projections to GnRH neurons (19), and a potential site at which cross-talk between the NMDAR and ER could be mediated.
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Materials and Methods |
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Surgical procedure
The rats were ovariectomized bilaterally under isoflurane anesthesia and allowed to recover for 2–3 wk. Six of the rats were implanted with estrogen (10% estradiol benzoate powder, 90% cholesterol powder) and the other five given cholesterol (control) in 1-cm SILASTIC-brand tubes (Dow Corning, Midland, MI) (inner diameter 1.96 mm, outer diameter 3.18 mm). Previous studies from our laboratory using the identical OVX + estrogen-replacement protocol indicate that this treatment results in approximately physiological levels of estrogen, similar to those in proestrous rats (23, 24). These capsules were implanted sc in the rats under isoflurane anesthesia. Two days later, the animals were deeply anesthetized with 0.35–0.5 ml of ketamine (100 mg/ml) and 0.35–0.5 ml of xylazine (20 mg/ml). The rats were perfused initially with 1% paraformaldehyde (50 ml) at a rate of 50 ml/min, followed by 4% paraformaldehyde (500 ml). The brains were removed and postfixed for 4–6 h in 4% paraformaldehyde, and then transferred into PBS with 0.1% sodium azide. Tissue sections (40 µm) were cut on a vibratome (Ted Pella, Redding, CA) and stored in PBS with 0.1% azide.
Fluorescence immunocytochemistry
Sections were washed in PBS and preblocked for 1 h with 10% normal goat serum (NGS) and 10% normal horse serum (NHS). Sections were then placed in a solution of the two primary antibodies, the rabbit polyclonal antibody to ER (1:5000, C1355, Upstate Biotechnology, Inc., Lake Placid, NY), which recognizes both bound and unbound ER (25) and the mouse monoclonal antibody to the NMDA-R1 subunit (54.1, 5 µg/ml dilution) (kindly provided by Dr. John H. Morrison) for 4 d, and stored on a rotator at 4 C. Both of these antibodies have been extensively validated and are highly specific (25, 26, 27). After the primary antibody incubation, sections were again washed and then incubated in a combination of the two fluorescent secondary antibodies (1:400 dilution of fluorescein isothiocyanate antirabbit IgG for ER and Texas Red antimouse IgG for NR1, Vector Laboratories, Burlingame, CA) for 1.5 h. After incubation, sections were washed, mounted on gelatin-coated slides, and stored in the dark. Slides were allowed to dry overnight and then coverslipped with Vectashield (Vector Laboratories). In other experiments, we incubated the tissues in the antibodies sequentially rather than simultaneously, and identical results were obtained by the two methods. In control experiments, the primary antibody was omitted to ensure that there was no nonspecific binding of the secondary antibody.
Fluorescence microscopy and analyses
Sections were examined at a magnification of x100 on a Zeiss laser scanning microscope 410 inverted confocal microscope (Carl Zeiss, Jena, Germany) for qualitative analysis of ER and NR1 double labeling. A suitable contrast/brightness setting that yielded a high-resolution image for the cells was determined and used to produce the images. The stored images then were transferred to Adobe Photoshop and printed with a Fujix Pictrography 3000 printer (Prographics, New York, NY).
Immunocytochemistry for stereological analysis
Four to five sections were taken for each region per animal at alternate intervals and were rinsed in buffer at room temperature on a shaker. Then, the sections were treated to eliminate any endogenous peroxide activity (3:1 methanol: %H2O2, 20 min at room temperature). Sections were washed, then incubated in the rabbit polyclonal ER antibody (1:5000, C1355, Upstate Biotechnology, Inc.) in 10% NGS and 10% NHS for 3 d at 4 C on a shaker. Then, the sections were rinsed and incubated in biotinylated antirabbit IgG (1:300, Vector Laboratories) for 1 h followed by rinsing in PBS. After rinsing, sections were incubated in avidin-biotin-peroxidase complex (Vector Laboratories) for 1 h. They were rinsed in buffer and developed in 3,3'-diaminobenzidine/peroxidase reaction. Then, sections were rinsed in PBS, and incubated in primary antibody to NR1 (54.1, 5 µg/ml dilution), in 10% NGS and 10% NHS for 3 d at 4 C on a shaker. The validation of these two antibodies is described above. After 3 d, they were washed and incubated in biotinylated antimouse IgG (1:300, Vector Laboratories) for 1 h. Sections were then incubated in avidin-biotin-peroxidase complex (Vector Laboratories) for 1 h. After a series of washes they were developed in VIP/peroxidase reaction (Vector VIP substrate kit, Vector Laboratories). Sections were rinsed, dried at room temperature and dehydrated in graded alcohols series, stained with cresyl violet, and coverslipped with DPX.
Stereological analysis
Cresyl violet-stained sections were studied quantitatively using standard stereological procedures for light microscopy (28, 29). The regions of the AVPV and MPN were drawn with the help of a rat brain atlas (30). Quantitative analyses were performed on the AVPV or MPN on one side of the tissue. Using a computer-assisted morphometry system consisting of a Zeiss Axioplan 2 photomicroscope (Carl Zeiss) equipped with an Applied Scientific Instrumentation (Eugene, OR) MS-2000XYZ computer-controlled motorized stage, a DAGE-MTI (Michigan City, IN) DC 330 video camera, a Dell (Austin, TX) microcomputer, and MicroBrightField (Colchester, VT) morphometry and stereology software. Stereologic methods using the optical fractionator protocols in MicroBrightField were used for all analyses. Estimates of ER immunoreactive nuclei and ER/NR1+-immunoreactive double-labeled cells were obtained using the optical fractionator. In a few cases, it was difficult to decide whether a cell was double or single labeled; although they were identifiable as ER-positive, we could not determine whether the cytoplasmic membrane was NR1 positive. These cells were counted separately in an uncertain (UC) group and were not used in the final determination of the percentages of single- or double-labeled ER-immunoreactive cells.
For stereological analyses, each cresyl violet counterstained, immunolabeled section was first viewed at low magnification using a 10x objective and each region (AVPV and MPN) was outlined on the live computer image. The stereoInvestigator software placed disector frames using a systematic-random design within each contour outlining each region. A 63x immersion oil, 1.4 numerical aperture objective was used to achieve optimal optical sectioning during disector analysis.
In the regions of AVPV and MPN, a 100 x 100 µm square grid was placed in a systematic random fashion over each region and the DAB stained for ER single-labeled cells, the ER/NR1+ double-labeled cells and UC cells were counted within a 50 x 50 µm optical disector in the x-y-axis. The final post-processing thickness of the sections was measured by the microcator. Because the mounted section thickness was on average 10 µm, the counting frame height was kept at 6 µm for all sections studied. The numbers of ER/NR1+ double-labeled cells, ER single-labeled cells (i.e. no NR1) and the UC neurons that fell within the disector frames were counted as separate populations.
Statistical analysis
Differences in ER/NR1+, ER/NR1-, or UC cells in the regions of AVPV and MPN were compared between the cholesterol and estrogen treated animals. The number of neurons counted from the StereoInvestigator program was calculated according to West et al. (31). In each case, one-way ANOVA was performed for effects of estrogen vs. vehicle. Significance was set at P < 0.05.
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Results |
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and NR1 immunoreactive cells immunoreactive nuclei was highly concentrated in specific nuclei such as the AVPV and MPN, in which both NR1 and ER exhibited overlapping dense expression. Fluorescence microscopy demonstrated a substantial amount of colocalization of these two molecules in these regions (Fig. 1
, A–F) with most ER nuclei colocalized with NR1. Representative micrographs of ER-immunoreactive nuclei are shown in green (A and D), NR1-immunoreactive cells are shown in red (B and E), and C and F are double-exposures demonstrating coexpression of ER and NR1 in the same cells. Therefore, we sought to quantify and to determine effects of estrogen on this colocalization, using unbiased stereological methods.
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and NR1 in the AVPV (dark brown nuclei) and NR1+ immunoreactive cells (lighter brown labeling of membranes on cytoplasm and processes), at the level of the AVPV is shown in Fig. 1H
. Higher power micrographs (x40 and x63) of single and double-labeled cells are shown in Fig. 1, I and J, respectively. The number of ER positive nuclei that were single-labeled or double-labeled with NR1 was counted using an unbiased stereological method and compared between OVX + vehicle and OVX + estrogen-treated young female rats. The percentage of ER nuclei colocalized with NR1 was 76% and 88% in vehicle and estrogen-treated rats, respectively, whereas 24% and 12% of ER-positive cells in the groups did not express NR1. Estrogen replacement caused a significant (47%) increase in the number of cells that coexpressed NR1 and ER in the AVPV (P < 0.05, Fig. 2). When the number of ER/NR1- cells was quantified in the AVPV, there was no significant difference in rats with estrogen compared with vehicle treatment (Fig. 3). The UC group represented a relatively small percentage of the cells, and there was no difference in UC cells between estrogen- and vehicle-treated rats in the AVPV. In cholesterol-treated rats, 55 ± 24 UC cells were counted, and in estrogen-treated rats, 35 ± 16 UC cells were counted.
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and NR1 in the MPN-immunoreactive cells (dark brown nuclei) and NR1-immunoreactive cells (light brown, localized on membranes of cytoplasm and processes) is shown in low power (x2.5) and high power (x40 and x63) at the level of the MPN in Fig. 1, L, M, and N, respectively. The number of ER nuclei that were single- or doubled-labeled with NR1 was counted using an unbiased stereological method in OVX + vehicle and OVX + estrogen-treated rats. The percent of ER nuclei that colocalized with NR1 in MPN was 81% and 84% in vehicle and estrogen treated rats, respectively.
As shown in Figs. 2 and 3, there were no significant differences in ER/NR1+ or ER/NR1- cells in the MPN region between the estrogen-treated and cholesterol-treated animals. The UC group again represented a relatively low percentage of the total ER population, and did not differ between estrogen and control rats in the MPN (cholesterol-treated rats, 62 ± 25; estrogen-treated rats, 38 ± 13).
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Discussion |
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and NR1 are substantially colocalized colocalizes with NR1, the NMDA receptor subunit that is obligatory to form a functional NMDAR (32). Using quantitative techniques in two selected brain regions, we found that there was extremely high colocalization of these receptors in the AVPV (76% and 88% of ER cells colocalize with NR1 in vehicle- and estrogen-treated rats, respectively) and the MPN (81% and 84% coexpression in control and estrogen-treated rats, respectively). This result leads us to speculate that this anatomical relationship may underlie the synergism of glutamate with estrogen in their physiological effects on GnRH and other neuroendocrine and autonomic systems. This anatomical relationship of NR1 with the ER has not been reported previously, although other studies are consistent with our finding. One group demonstrated the presence of ER mRNA in cells expressing the NMDAR2d subunit mRNA using RT-PCR in hypothalamus (33). However, no regional specificity could be ascertained from that study as cells were taken throughout the hypothalamus. Approximately 50% of ER cells in the MPN express non-NMDA (AMPA) glutamatergic receptors (34). If the ER can colocalize with both NMDA and non-NMDA receptors in the same cell, this could have functional implications, as the ability of NMDA receptors to be activated by glutamate is facilitated by prior depolarization of the cell by a non-NMDA or other ionotropic receptor (35). Our present finding that between 81% and 84% of cells in the MPN expressing ER also express the NMDAR subunit, depending upon ovarian hormonal status, and that approximately 50% of ER cells express non-NMDARs in the MPN in the study by Diano et al. (34) also suggests a high likelihood that the same cells can express ER, NMDARs and non-NMDARs. Taken together, these results indicate that estrogen and glutamate (through the NMDAR and/or non-NMDAR) can interact directly within the same target cells in the hypothalamus.
Effects of estrogen on ER and NR1 in AVPV
The AVPV is critically involved in the integration of the neural circuitry controlling gonadotropin secretion (20). It is sexually dimorphic and hormonally regulated. Many of the sex differences in the anatomy and functions in the AVPV are due to the differential exposure of males and females to testosterone and/or estradiol during prenatal development (36, 37, 38, 39). With respect to the GnRH neurosecretory system, the AVPV is necessary for the estrogen-induced preovulatory GnRH/LH surge, as lesions of this region obliterate the surge, and decrease fos expression in ipsilateral GnRH neurons during the surge (20, 40). These functions appear to involve glutamate as blockade of the NMDAR also reduces or attenuates the surge (11, 41), indicating an interaction between the ER and the NMDAR in the regulation of this crucial physiological function. In addition the AVPV of rats contains a high density of NMDARs, and both ER and ERß (42, 43), suggesting that it is an important site for the interaction of glutamate and sex steroid hormones.
In the present study, we performed a quantitative stereological analysis to determine the numbers and percentages of ER cells that coexpress NR1. This type of unbiased approach is important for these analyses because the distribution of ER and NR1 may not be homogeneous in the hypothalamus/preoptic area and even within a specific brain nucleus. Our results show that estrogen treatment to OVX animals causes an increase in the number of cells that coexpress ER and NR1, from 76–88% in the AVPV. However, ER cells that do not express NR1 are unaffected by estrogen, suggesting that effects of estrogen are specific to the subpopulation of ER cells that also express NR1, although we do not know the mechanism for this effect. This finding of high colocalization of ER and NR1 and its regulation by estrogen suggests that the signaling pathway of estrogen through its nuclear ER can interact with glutamate signaling through the NMDAR to have influences on downstream targets.
The present results showing an increase in coexpression of ER and NR1 in the AVPV might not be predicted by data from the laboratory of Simerly (42). They showed that estrogen treatment for 2 d to juvenile female rats caused a down-regulation of NR1 mRNA levels in the AVPV. This might be expected to result in a decrease in the number of ER cells expressing NR1 following estrogen treatment. However, that report differed from ours in several ways, including the age of the animal, the time after OVX when estrogen was administered, and the endpoint [mRNA in the case of the study by Gu et al. (42) and protein in our present report. It is quite possible that changes in NR1 mRNA levels may not be reflected by similar changes in protein, and indeed, our laboratory has shown an uncoupling of mRNA and protein levels of this molecule in the hypothalamus (44). In addition, and perhaps most importantly, our study specifically focused only on those NR1-positive cells that were coexpressed with ER. Other NR1 cells that do not express ER might have differential estrogen sensitivity (e.g. through ERß or nongenomic ERs, or through other neurons that express ERs).
Effects of estrogen on ER and NR1 in MPN
The MPN is another sexually dimorphic nucleus (45). It is important for the mediation of sex-specific reproductive behavior (46). During the late prenatal and early postnatal periods of development the size of the MPN is organized by sex steroid hormones (47). These effects are manifested as differences in the size of the MPN as well as the size and the number of neurons in this region.
Using stereological analysis, we observed extensive colocalization of ER and NR1 in the MPN, similar to our findings in the AVPV. However, quantitative experiments showed that there were no significant differences in the colocalization of ER and NR1 in MPN in estrogen compared with vehicle-treated rats (84% vs. 81%, respectively). Other studies have reported that the concentration and number of ER in the preoptic area decrease after estrogen treatment (48, 49) and ER mRNA levels are also reduced following estrogen treatment (50). In our study, there was a slightly decreased number of all ER-positive cells (both those that express NR1, as well as those that do not express NR1) in the MPN of estrogen-treated rats compared with vehicle controls, although this did not attain significance.
Regional differences in estrogen responsiveness, and implications for GnRH neurons
ER and NR1 are both extensively colocalized in the AVPV and MPN. However, there is a distinct regional specificity in the response to estrogen on the percentage of the cells that are colocalized. In AVPV, the number of ER nuclei colocalized with NR1 was significantly greater in estrogen treated animals than control rats. In MPN no significant effects of estrogen on colocalization were seen. These results indicate the importance of performing such analyses in specific regions, as effects of estrogen can vary substantially among different preoptic or hypothalamic nuclei.
The robust coexpression of ER and NR1 in a high percentage of cells and its regulation by estrogen has implications for the GnRH system. GnRH neurons respond to fluctuating levels of estrogen throughout the ovarian cycle with changes in cyclic patterns of biosynthetic and electrical activity (reviewed in Ref. 51). Estrogen positive and negative feedback in the brain of female vertebrates via nuclear ERs ( and ß) plays a critical role in the regulation of reproductive functions. Although GnRH cells probably do not express appreciable amounts of ER (4, 5, 6), this receptor is clearly implicated in the regulation of GnRH neural functions, albeit probably by indirect neurotransmission that could arise from other hypothalamic and preoptic regions, including the AVPV and MPN. Results showing potentiating effects of estrogen on mediating the NMDAR stimulation of GnRH cells (12, 16, 17) indicate that the NMDAR in these same brain regions is a possible target for the modulation of estrogen’s action on GnRH neurons.
In conclusion, our findings indicate an extensive colocalization of ER and NR1 in two sexually dimorphic brain regions involved in reproductive function: the AVPV and MPN. The coexpression of these receptors suggests that neural signals from estrogen and glutamate might be integrated at this cellular level. These findings have implications not only for GnRH neurons but potentially for other types of neuroendocrine and autonomic cells, many of which are also estrogen and glutamate sensitive.
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Acknowledgments |
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Footnotes |
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Abbreviations: AVPV, Anteroventral periventricular nucleus; ER, estrogen receptor; MPN, medial preoptic nucleus; NGS, normal goat serum; NHS, normal horse serum; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; NR1, obligatory NMDAR subunit; OVX, ovariectomized; UC, uncertain.
Received July 23, 2002.
Accepted for publication September 12, 2002.
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-containing neurons projecting to the vicinity of the gonadotropin-releasing hormone perikarya in the rostral preoptic area of the rat. J Comp Neurol 411:346–358[CrossRef][Medline]
and ß. J Biol Chem 275:7158–7166 immunoreactivity in the preoptic brain, the diencephalon, and the amygdala in the rat. J Comp Neurol 389:81–93[CrossRef][Medline]
-hydroxyestrone on pituitary and preoptic estrogen receptor regulation. Endocrinology 125:2701–2709This article has been cited by other articles:
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J. H. Morrison, R. D. Brinton, P. J. Schmidt, and A. C. Gore Estrogen, Menopause, and the Aging Brain: How Basic Neuroscience Can Inform Hormone Therapy in Women J. Neurosci., October 11, 2006; 26(41): 10332 - 10348. [Full Text] [PDF]
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, P < 0.05, significant difference between estrogen-treated vs. corresponding control. CHOL, Cholesterol-treated (vehicle) rats.