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Department of Obstetrics and Gynecology and Center for Research in Reproductive Biology, Yale University, New Haven, Connecticut 06520
Address all correspondence and requests for reprints to: Tamas L. Horvath, Yale Medical School, Department of Obstetrics and Gynecology, 333 Cedar Street, FMB 339, New Haven Connecticut, 06520. E-mail: HorvathTA{at}MASPO1.MAS.YALE.EDU
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Abstract |
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-amino-3-hydroxy-5-methyl-4-isoxazone-propionate
(AMPA) GluR1, GluR2/3, and androgen receptors or estrogen receptors was
revealed in the same cells of septal, amygdaloid, and hypothalamic
areas by double immunocytochemistry. The highest incidence of
colocalization was detected in hypothalamic regions. To demonstrate a
regulatory role of testosterone or estradiol on AMPA receptor
expression, the hormonal milieu of male and female rats was manipulated
by gonadectomy and hormonal treatment. GluR1 and GluR2/3 expression was
assessed by Western blots. Statistical analysis demonstrated that
testosterone and estradiol have a stimulatory influence on the
expression of AMPA receptors in the hypothalamus. The regulatory effect
of estradiol on AMPA receptors was found to be site and gender
specific: after estradiol treatment, samples taken from the
hypothalamus contained increased levels of GluR1 and GluR2/3, whereas
in the septum, bed nucleus and amygdala, no changes could be detected.
Furthermore, the increase in hypothalamic GluR 2/3 levels was two times
higher in females, compared with males, whereas the changes in
hypothalamic GluR 1 levels showed no sex differences. Our results support the hypothesis that the interaction between gonadal steroids and glutamate involves hormone regulation of GluR. This mechanism seems to be gender and site specific, suggesting that excitatory neurotransmission and related physiological mechanisms also may be distinctly different in males and females.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsExp 1ResultsDiscussionReferences |
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-amino-3-hydroxy-5-methyl-4-isoxazone propionic
acid), kainate, and NMDA (N-methyl-D-aspartate)
receptors. The activation of ionotropic receptor-gated channel-complex
results in Ca2+ increase, which may regulate neuronal excitability
(10), cell differentiation (11), long-term potentiation (12), and
stimulation of peptide biosynthesis (13). Synergistic interactions between glutamate and gonadal steroids may underlie various limbic and hypothalamic functions (for review, see 9). For example, administration of estradiol and progesterone induces an increase in glutamate binding sites in the hypothalamus of female rats (14). The rate of glutamate release is increased also in the medial preoptic area during an estrogen-induced LH surge (15). It is suggested that the elevation of glutamate binding sites and glutamate action in these areas are, at least in part, associated with AMPA receptor levels (16, 17, 18). To support the role of AMPA receptors in these mechanisms, in situ hybridization (7) and immunocytochemical studies (5) showed that two particular subunits of AMPA GluR (GluR1 and GluR2) are widely distributed in hypothalamic and limbic areas of female and male rats where gonadal steroid action is anticipated. However, the hypothalamic and limbic sites, where hormonal signals may be integrated into excitatory neural circuits, are yet to be defined.
We hypothesize that hormonal signals may affect excitatory neurotransmission, at least in part, by regulating the expression of AMPA GluR in the septum, amygdala, and hypothalamus, where these receptors coexist with gonadal steroid binding sites. Because developmental sexual dimorphism exists in hypothalamic areas, we propose also that hormonal regulation of AMPA receptors may occur in a gender-specific manner. To test these hypotheses, first we determined hypothalamic and limbic neuronal populations that are targets of both glutamate and gonadal steroid actions using immunolabeling for AMPA- and gonadal steroid receptors. AMPA receptors are present exclusively in the cytoplasm and perikaryal membrane (5, 19, 20), whereas the gonadal steroid receptors are restricted to the nucleus of cells (21, 22, 23), allowing easy detection of the two tissue antigens in the same cell. The second objective of this study was to determine whether, in sites where coexistence of GluR and steroid receptors is revealed, gonadal hormones regulate AMPA receptor levels, and whether this mechanism is sexually dimorphic. This hypothesis was tested by manipulating the hormonal environment of male and female rats, and subsequently, Western blot analyses were used to assess the AMPA receptor content of different limbic and hypothalamic regions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsExp 1ResultsDiscussionReferences |
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Exp 1 |
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Immunostaining.
Because of the different subcellular
compartmentalization of GluR (cytoplasm, perikaryal membrane) and
gonadal steroid receptors (nucleus), the light microscopic double
immunostaining for GluR1 or GluR2/3 and androgen receptors (AR) or
estrogen receptors (ER) was carried out according to the following
protocol: Incubation in antisera generated in rabbit against either
GluR1 or GluR2/3 (Chemicon, Temecula, CA) diluted 1:500 in PB
containing 0.3% Triton X-100 for 24 h at room temperature. After
several washes in PB, sections were incubated in the secondary antibody
(biotinylated goat antirabbit IgG, 1:250 in PB containing 0.3% Triton
X-100; Vector Laboratories, Burlingame, CA) for 2 h at room
temperature, then rinsed in PB 3 x 10 min. This was followed by
incubation in avidin-biotin peroxidase (1:250 in PB, ABC Elite, Vector
Labs) for 2 h at room temperature, and the tissue-bound peroxidase
was visualized with a nickel-diaminobenzidine (Ni-DAB) reaction (15 mg
DAB, 0.12 mg glucose oxidase, 12 mg ammonium chloride, 600 µl of 0.05
M Ni ammonium sulfate, 600 µl 10%
ß-D-glucose in 30 ml PB; dark blue reaction product in the
cytoplasm). After several rinses in PB, sections were further
immunostained for either AR or ER. After a 48-h (at 4 C), incubation in
either rabbit anti-AR (2 µg/ml; gift from Dr. Gail Prins, University
of Illinois, Chicago, IL; 23 or rabbit anti-ER (1:500; National
Hormone and Pituitary Program; Refs. 21, 22), sections were further
processed using the peroxidase-antiperoxidase technique: incubation in
the secondary antibody (goat-antirabbit IgG), 1:50 in PB for 2h at room
temperature, followed by rabbit peroxidase-antiperoxidase, 1:100 in PB.
Between incubation steps, sections were rinsed 3 x 15 min in PB.
The tissue bound peroxidase was visualized by a light brown DAB
reaction (15 mg DAB, 165 µl 0.3% H2O2 in 30
ml PB, 5–10 min at room temperature; brown reaction product in the
nucleus). After immunostaining of the second tissue antigen, sections
were thoroughly rinsed in PB, placed on gelatin coated slides,
dehydrated through increasing ethanol concentrations, and mounted with
Permount. In control double-immunostaining experiments, one or both of
the primary antibodies were replaced with normal serum. Either single
or no immunostaining was detected under these circumstances.
For electron microscopic studies, double immunostaining for GluR1-2/3 and AR or ER was carried out according to the following protocol: Incubation was performed with a mixture of rabbit anti-AR (2 µg/ml; gift from Dr. Gail Prins, University of Illinois, Chicago, IL; 23 or rabbit anti-ER (1:500; National Hormone and Pituitary Program; Refs. 21, 22) and antisera generated against either rabbit anti-GluR1 or GluR2/3 (Chemicon) diluted 1:500 in PB for 24 h at room temperature. After several washes in PB, sections were further incubated in the secondary antibody (biotinylated goat antirabbit IgG, 1:250 in PB; Vector Labs) for 2 h at room temperature, then rinsed 3 x 10 min. This was followed by incubation in ABC Elite (Vector Labs) for 2 h at room temperature, and the tissue-bound peroxidase was visualized with a Ni-DAB reaction (dark blue reaction product). Subsequently, sections were postosmicated (1% OsO4 in PB) for 30 min, dehydrated through increasing ethanol concentrations (using 1% uranyl acetate in the 70% ethanol for 30 min), and flat embedded in araldite between liquid release-coated slides (Electron Microscopy Sciences, Fort Washington, PA), and placed in an oven at 60 C for 48 h. After capsule embedding, blocks were trimmed and cut on a Reichert-Jung ultramicrotome. Ribbons of ultrathin sections were collected on Formvar-coated single-slot grids and examined using a Philips CM-10 electron microscope (Mahway, NJ).
Exp 2
Hormonal manipulations.
Male (n = 6) and female (n
= 6) rats were left intact or bilaterally ovariectomized (n = 12)
or castrated (n = 16) under ether anesthesia. Four weeks later,
groups of gonadectomized females (n = 6) and males (n = 4)
received daily sc injections of 17-ß-estradiol benzoate (10 µg/100
g BW diluted in sesame oil) for 7 days (24). A group of castrated males
(n = 6) were sc injected daily with testosterone (500 µg/100 g
BW in sesame oil) for 7 days (24). The remainder of the gonadectomized
animals (6 males and 6 females) received oil injection alone.
Western blot analysis.
Rats from each group were
killed by decapitation 24 h after the last injection to the
hormone-treated animals. The hypothalamus, septum, and amygdala of each
animal were removed and homogenized in lysis buffer containing 50
mM Tris-HCl (pH 7.5), 50 mM MgCl2,
5 mM EGTA, 0.25% Triton X-100, and protease inhibitors
(proteinase inhibitor cocktail tablets-Boehringer Mannheim,
Indianapolis, IN) for the protein isolation. The homogenated tissues
were centrifuged at 190,000 x g for 1 h at 4 C.
The resulting supernatant was normalized for total protein using the
bicinchonic acid assay (BCA Protein Assay, Pierce Rockford, IL).
Coomassie-stained SDS-polyacrylamide gels were routinely used to
evaluate the concentration and quality of the extracts.
Western blots were carried out using 10% SDS-polyacrylamide gels run on a minigel apparatus; 30 µg of protein was loaded per lane. The gels were transferred to polyvinylidene fluoride (Millipore Corp. Bedford, MA) membranes by electroblotting overnight (30 V). The filters were blocked in 5% nonfat dry milk and 0.1% Tween for 1 h at room temperature. Blots were then incubated with rabbit anti-GluR1 (0.5 µg/ml) or GluR2/3 (0.25 µg/ml) (Chemicon) diluted in TBS Tween (20 mM Tris, 137 mM NaCl, pH 7.6) for 1 h at room temperature. Blots were incubated also with monoclonal mouse anti-ß tubulin (1:15,000) (Sigma Chemical Co. St. Louis, MO) as controls to evaluate the amount of protein loaded for each lane. Membranes were washed three times for 10 min in the same buffer and incubated for 1 h with horseradish peroxidase-conjugated goat antirabbit IgG or horse antimouse IgG (Vector Labs) diluted 1:15,000 in TBS Tween. Subsequently, the blots were washed five times for 10 min in the same buffer. Immunoreactive proteins were revealed using enhanced chemiluminescence method (ECL, Amersham, Arlington Heights, IL).
Data analysis
The extent of colocalization of GluR1 or GluR2/3 and ER or AR
was calculated by quantitative analysis. Double-immunolabeled vibratome
sections from five male and five female rats were examined under the
light microscope. The number of single- and double-labeled neurons was
counted in each area of interest. The percentage of gonadal steroid
receptor-immunoreactive cells (immunolabeled nuclei) that were
immunopositive for AMPA receptors (immunolabeled perikarya) and the
percentage of AMPA receptor-containing cells that were immunoreactive
for gonadal steroid receptors were calculated in each region.
The expressions of GluR1 and GluR2/3 on Western blot analysis were quantified densitometrically using an image analysis system (ImageQuantNT software; Molecular Dynamics, Sunnyvale, CA). Each gel contained samples from each region studied in gonadectomized and gonadectomized plus hormone-treated animals. The value of each sample was normalized with the value of the corresponding ß tubulin. Then, the values of samples taken from gonadectomized animals were considered to be at the 100% level of protein. Thus, the values of GluR1 and GluR2/3 levels of intact and steroid-treated animals are given in arbitrary densitometric units.
Differences were assessed between groups using the ANOVA. All data are presented as the mean ± SEM. Statistical significance was assumed for P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsExp 1ResultsDiscussionReferences |
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-5). Table 1 shows the percentage of
colocalization of AMPA receptors and gonadal steroid receptors in areas
of the limbic system and hypothalamus.
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Immunostaining for GluR2/3 resulted in a distribution pattern of labeled neurons similar to that of GluR1. However, in the lateral septum, the subcellular distribution of GluR2/3 immunoreactivity seemed to be different from that of GluR1. Here, labeling was predominantly in the soma, whereas only the proximal dendrites were lightly stained.
Electron microscopy. In most cells of all regions,
immunolabeling was most intense in the cell membrane with weaker,
patchy labeling throughout the cytoplasm of both somata and dendrites
(Figs. 2 and 3). No labeling was detected
in axons, axon terminals, nuclei, or nucleoli.
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and 5). Fewer AR-immunoreactive nuclei were detected in the
diagonal band of Broca and other hypothalamic nuclei. The distribution
pattern of ER-immunoreactive perikarya overlapped that of the AR,
except for the dorsal divisions of the lateral septum. In this latter
region, no ER positive cells were found.
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Exp 2
GluR1 and GluR2/3 levels in intact and castrated plus
testosterone-treated males, as compared with castrated controls, using
Western blot analysis (Figs. 6 and 7).
Compared with castrated males (n = 6), animals with
testosterone treatment (n = 6) showed a significant
(P < 0.05) increase in GluR1 protein levels in the
hypothalamus (41% ± 8.54) (Figs. 6A and 7A). GluR2/3 also was
increased by 33% ± 21.3 in the hypothalamus of testosterone-treated
castrated animals (Figs. 6B and 7B). Changes (
) in either of these
AMPA receptor levels could not be detected in the septum or in the
amygdala (Figs. 6 and 7).
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and 7A) and GluR2/3 was higher
by 17% in the hypothalamus (Figs. 6B and 7B). Samples taken from the
septum and amygdala of intact and castrated control animals did not
show differences (Figs. 6 and 7).
GluR1 and GluR2/3 levels in castrated plus estradiol
benzoate-treated males, as compared with castrated controls, using
Western blot analysis (Figs. 6 and 7).
Treatment of castrated male
rats (n = 4) with estradiol benzoate increased the amount of GluR1
by 53% ± 6.88 in the hypothalamus, compared with castrated controls
(n = 4; Figs. 6A and 7A). The amount of GluR2/3 in treated animals
also was increased by 29% ± 1.5 in the hypothalamus (Figs. 6B and 7B). in GluR 1 or GluR2/3 levels could not be detected in the
septum or in the amygdala (Figs. 6 and 7).
GluR1 and GluR2/3 levels in intact females and ovariectomized plus
estradiol benzoate-treated females, as compared with ovariectomized
controls, using Western blot analysis (Figs. 8 and 9).
Compared with ovariectomized females (n =
6), ovariectomized plus estradiol-benzoate-treated animals (n =
6), increased their GluR1 and GluR2/3 protein content in the
hypothalamus. This increase for GluR1 was 43% ± 13.63 (Figs. 8A and 9A), whereas for GluR2/3 level, was 55% ± 5.9 (Figs. 8B and 9B).
in either of these AMPA receptor levels could not be detected in the
septum or in the amygdala (Figs. 8 and 9).
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and 9). This may reflect that intact females were most
probably killed at days of their estrus cycle when estrogen levels were
low.
Male-female differences in the of GluR2/3 levels after
estradiol benzoate treatment (Fig. 10).
Western
blot analysis showed increased expressions of AMPA GluR1 and GluR2/3 in
gonadectomized female and male rats after estradiol treatment (see
above). In the hypothalamus, GluR2/3 was about 2-fold higher
(P < 0.05) in females (55% ± 5.9), compared with
males (29% ± 1.5; Fig. 10). On the other hand, no significant
differences could be detected between GluR1 of males (53% ± 6.88)
and that of females (43% ± 13.63; Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsExp 1ResultsDiscussionReferences |
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The experimental design of this study was based on the premise that gonadal steroids exert their effect through classical, nuclear receptors. In fact, the colocalization studies indicate that the effects of estradiol and testosterone on the expression of AMPA receptors may be mediated by estrogen or AR: the area that showed significant increases in GluR1-3 expression, the hypothalamus, had the highest incidence of colocalization of glutamate and gonadal steroid receptors.
Functional implications
Hypothalamus.
The detection of gonadal steroid receptors in
hypothalamic neurons expressing different subtypes of AMPA receptors
and the upregulation of these GluR by gonadal steroids may have
implications for a possible mechanism in the central regulation of
neuroendocrine processes.
An increasing body of data indicates that glutamate plays a role in the hypothalamic regulation of pituitary hormones, including ACTH, GH, PRL, oxytocin, vasopressin, and gonadotropins (28, 29, 30, 31, 32, 33, 34, 35). For example, endogenous excitatory amino acids induce gonadotropin secretion from the anterior pituitary during the ovarian cycle and under experimental conditions (36, 37, 38). The effect of glutamate on gonadotropin secretion seems to be mediated by the hypothalamus, because: 1) direct administration of glutamate to the pituitary gland does not alter gonadotropin secretion (39); 2) glutamate-induced LH secretion can be blocked by the administration of an LH-releasing hormone (LHRH) antagonist (40). Supporting a central site of action, Bourguignon et al. (17) have reported that incubation of rat hypothalami in vitro with glutamate analogs results in a dose-related stimulation of LHRH release. Donoso et al. (41) and Abbud and Smith (42) have found that in vitro, non-NMDA receptors are more potent in stimulating LHRH release from arcuate nucleus and median eminence (ME fragments). Administration of kainic acid or quisqualic acid readily increased the release of LHRH, whereas NMDA injected in physiological concentrations was ineffective. Furthermore, the glutamate-induced release of LHRH from arcuate nucleus/ME fragments could be blocked by the AMPA/kainate receptor antagonist, DNQX, but not by the NMDA receptor antagonist, AP-7 (41). Moreover, Brann et al. (43) have demonstrated that castration does not affect NMDA receptor concentrations in the hypothalamus of female and male rat brain. Steroid replacement to ovariectomized and castrated rats does not have an effect on NMDA receptors binding in the hypothalamus or cerebral cortex (43). On the other hand, Weiland (14) found an increase in [3H] glutamate binding in the preoptic area of ovariectomized rats after estradiol plus progesterone treatment. In light of the views that estradiol (44) and glutamate (42, 45, 46) exert their effects on LHRH neurons, at least in part indirectly, we propose that the actions of glutamate and estradiol on gonadotropin secretion are integrated in LHRH-targeting hypothalamic neurons that coexpress gonadal steroid receptors and AMPA receptors. Furthermore, we suggest that estradiol could amplify the effect of glutamate on LHRH by increasing the expression of different AMPA receptor subunits in hypothalamic nuclei. Further studies are needed to determine whether changes in AMPA receptors content occur in female rats during the estrus cycle under different estrogen environment. Moreover, it is necessary to assess whether the increased expression of AMPA receptors occurred in neurons with ER, and if so, whether ER/AMPA containing neurons have direct efferents or access (for example, via nitric oxide) to LHRH cells.
Amygdala, bed nucleus of stria terminalis, septum.
The
amygdala, bed nucleus of stria terminalis, and septal area are
conspicuous, integrated parts of the limbic system. Through extensive
and reciprocal interconnections with the limbic telencephalic and
diencephalic areas and with mesencephalic, lower brain stem and spinal
cord regions, these areas are involved in the control of a variety of
physiological and behavioral processes related to higher cognitive
functions (2). These include emotion and memory, social behavior such
as reproduction and aggression, and modulation of the autonomic and
neuroendocrine system.
The medial nucleus of amygdala, in conjunction with the posteromedial bed nucleus and medial preoptic area, plays a major role in the integration of sensory and hormonal cues in the regulation of sexual behaviors (47, 48). The abundance of steroid receptors in these areas was described earlier (21, 49), and the present study is in absolute agreement with those descriptions. Our observation that approximately 50% of medial amygdala, bed nucleus, and medial preoptic ER/AR-containing neurons coexpress AMPA GluR raises the possibility that, in the chemosensory pathway, glutamate is a dominant neurotransmitter.
The septal area, particularly the lateral septum, is rich in glutamate and aspartate because of its massive innervation by excitatory amino acid-containing limbic cortical efferents (1, 2, 3). Substantial populations of neurons in this region also are direct targets of circulating gonadal steroids, including estradiol (49) and testosterone (23). Because the lateral septum is considered to be a site that relays hippocampal signals to hypothalamic regions that participate in gonadal steroid dependent mechanisms, such as gonadotropin control (medial preoptic area, arcuate nucleus) and sexual behaviors (ventromedial nucleus, medial preoptic area), it is surprising that we found very limited colocalization of gonadal steroid and AMPA GluR (10%) in septal neurons and no significant alteration of AMPA receptor expression after hormone treatment. Whereas technical limitations (antisera, sampling for the Western blots) may have contributed to these results, one may suggest that hippocampal and gonadal signals are not integrated at the level of the septum, but rather in the hypothalamus or in the hippocampus.
Conclusions
The present observations suggest that glutamate regulated neurons
in the hypothalamus are direct targets of gonadal steroids and that, by
altering the expression of AMPA GluR, androgen and estrogen may readily
influence excitatory neurotransmission in these areas in a sexually
dimorphic manner.
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Acknowledgments |
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Footnotes |
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Received August 13, 1996.
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, GluR2/3 immunolabeled
neurons; 
, GluR1-immunolabeled neurons; •, AR/ER immunolabeled
nuclei; 
, AR-immunolabeled nuclei. Overlapping images
indicate colocalization. cc, Corpus callosum; LS, lateral septum; BST,
bed nucleus of the stria terminalis; ac, anterior commissure; DBB,
diagonal band of Broca; acp, anterior comissure, posterior; MPN, medial
preoptic nucleus; oc, optic chiasm; fx, fornix; VMN, ventromedial
hypothalamic nucleus; ARC, arcuate nucleus; III, third ventricle; ME,
median eminence; CeA, central amygdaloid nucleus; MeA, medial
amygdaloid nucleus; opt, optic tract; D3V, third ventricle, dorsal.
