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The Journal of Neuroscience, April 15, 1999, 19(8):3213-3222
Hormonal Regulation of Glutamate Receptor Gene Expression in the
Anteroventral Periventricular Nucleus of the Hypothalamus
Guibao
Gu1,
Frederique
Varoqueaux1, and
Richard B.
Simerly1, 2
1 Division of Neuroscience, Oregon Regional Primate
Research Center, Beaverton, Oregon 97006, and 2 Program in
Neuroscience, Oregon Health Sciences University, Portland, Oregon
97201
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ABSTRACT |
Glutamate plays an important role in mediating the positive
feedback effects of ovarian steroids on gonadotropin secretion, and the
preoptic region of the hypothalamus is a likely site of action of
glutamate. The anteroventral periventricular nucleus (AVPV) of the
preoptic region is an essential part of neural pathways mediating
hormonal feedback on gonadotropin secretion, and it appears to provide
direct inputs to gonadotropin releasing hormone (GnRH)-containing
neurons. Immunohistochemistry and in situ hybridization were used in this study to define the distribution and hormonal regulation of glutamate receptor subtypes in the AVPV of juvenile female rats. Neurons that express the NMDAR1 receptor subtype are
abundant in the AVPV, as are cells that express AMPA receptor subtypes
(GluR1, GluR2, and GluR3 but not GluR4), and the AVPV appears to
contain a dense plexus of NMDAR1-immunoreactive presynaptic terminals.
However, AVPV neurons do not seem to express detectable levels of
kainate receptor (GluR5, GluR6, and GluR7) or metabotropic receptor (mGluR1-6) subtypes. Treatment of ovariectomized juvenile rats with estradiol induced expression of GluR1 mRNA but did not alter
levels of GluR2 or GluR3 mRNA. Treatment of estrogen-primed ovariectomized juvenile rats with progesterone caused an initial increase in GluR1 mRNA expression, followed by a small decrease 24 hr
after treatment. In contrast, estrogen appears to suppress levels of
NMDAR1 mRNA in the AVPV, which remained unchanged after progesterone treatment. Thus, one mechanism whereby ovarian
steroids may provide positive feedback to GnRH neurons is by altering
the sensitivity of AVPV neurons to glutamatergic activation.
Key words:
preoptic region; gonadotropin releasing hormone; in
situ hybridization; ovarian steroids; progesterone; estrogen
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INTRODUCTION |
In both developing and mature
mammals, the preovulatory surge in luteinizing hormone (LH) secretion
is dependent on the activation of gonadotropin releasing hormone
(GnRH)-containing neurons in the preoptic region by estradiol and/or
progesterone, because these cells represent the final common pathway
for the neural control of ovulation (Gerall and Givon, 1992 ; Ojeda and
Urbanski, 1994). In the rat, GnRH neurons do not appear to express
estrogen or progesterone receptors (Shivers et al., 1983; Fox et al.,
1990), suggesting that these cells do not transduce the positive
feedback effects of ovarian steroids directly. However, it has been
known for some time that a neural signal originating from the preoptic area of the hypothalamus is required for the preovulatory LH surge (Halasz, 1969; Köves and Halász, 1970), and an emerging
body of evidence suggests that the anteroventral periventricular
nucleus (AVPV) in this area, which contains a high density of neurons expressing estrogen and progesterone receptors, plays a pivotal role in
mediating the positive feedback effect of ovarian steroids on
gonadotropin secretion (Wiegand and Terasawa, 1982; Simerly, 1996;
Herbison, 1998). Consistent with its proposed functional role, the AVPV
provides direct projections to a subpopulation of GnRH neurons in the
vascular organ of the lamina terminalis (OVLT) region (Gu and Simerly,
1997), which project to the median eminence and are thought to
participate in the initiation of the LH surge. However, it remains
unclear how AVPV neurons exert their stimulatory effects on the
activity of GnRH cells.
A variety of neurotransmitters have been implicated in the regulation
of the LH surge, and recent studies suggest that the activation of
receptors for glutamate plays a critical role in the
initiation of the steroid-induced LH surge that occurs during puberty and in adult animals. In immature and adult ovariectomized rats, the estrogen-induced LH surge can be blocked by administration of
an NMDA receptor antagonist or antagonists of both NMDA and non-NMDA receptors (Lopez et al., 1990; Urbanski and Ojeda,
1990). NMDA and non-NMDA antagonists can also block the
progesterone-induced LH surge in estrogen-primed ovariectomized
immature and adult rats (Brann and Mahesh, 1991a,b; Ping et al.,
1994a). On the other hand, the facilitatory effects of the activation
of AMPA and NMDA receptors appear to be dependent on estrogen (Arias et
al., 1993; Brann, 1995; Ping et al., 1995). At least some of these
effects occur at the level of the preoptic region because excitatory
amino acids (EAAs) stimulate GnRH release from preoptic tissue
fragments in vitro (Bourguignon et al., 1989), and injection
of EAAs into the preoptic region stimulates LH release (Ondo et al.,
1988). Therefore, glutamate receptors appear to represent essential
components in the neural transduction of hormonal positive feedback on
gonadotropin secretion. Moreover, sex steroid hormones may regulate the
expression or responsiveness of glutamate receptors in key neural
pathways or may alter glutamate release, thereby influencing the
expression and/or secretion of GnRH. To test this idea, we used
immunocytochemistry and in situ hybridization to determine
which subunits of glutamate receptors are expressed and hormonally
regulated in the AVPV of female rats.
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MATERIALS AND METHODS |
Animals and treatments. The protocols used here were
approved by the Oregon Regional Primate Research Center Institutional Committee for the Care and Use of Animals in Research and Education, in
accordance with the guidelines of the National Institute of Health and
United States Department of Agriculture. Juvenile female Sprague Dawley
rats were obtained from B & K Universal Inc. (Kent, WA) and housed on a
14:10 light/dark schedule with light on at 5:00 A.M. Food and
water were available ad libitum. Juvenile female Sprague
Dawley rats were ovariectomized bilaterally on day 22 of life and
implanted subcutaneously with a silastic capsule containing 17 -estradiol (E2) (400 µg/ml dissolved in corn oil; tubing
internal diameter, 1 mm; outer diameter, 2.16 mm; 20 mm in length/100
gm body weight; Sigma, St. Louis, MO) [n = 12 (4 for immunocytochemistry, 4 for in situ hybridization, and
4 for double in situ hybridization)] or control capsules
(oil only) [n = 12 (4 for immunocytochemistry, 4 for
in situ hybridization, and 4 for double in situ
hybridization)] 5 d later. This dosage of E2 has been confirmed
to reliably reproduce the preovulatory levels of plasma E2 measured
during the initiation of puberty in the rat (Andrews et al., 1981).
Forty-eight hours after implantation, paired groups of animals (treated
with E2 or control capsules) were killed by transcardial
perfusion. In addition, to simulate the sequence of events observed
during the afternoon of the first proestrus, a time during which
elevated E2 levels are accompanied by a marked and abrupt increase in
progesterone levels (Parker and Mahesh, 1976; Andrews et al., 1980),
four groups of animals (n = 4 for each group) were
ovariectomized and treated with E2 (see above) for 48 hr and then
injected subcutaneously with progesterone at 12:00 P.M. (1 mg/animal;
Sigma) or corn oil and perfused 3 or 24 hr after injection. This dosage
of progesterone can markedly increase GnRH mRNA levels in
estrogen-primed immature rats (Kim et al., 1989; Ma et al., 1992) but
not in animals without pretreatment with estrogen (Ma et al., 1992).
Two time points (3 and 24 hr) were evaluated to compare the short- and
long-term effects of progesterone on the expression of glutamate
receptors, which are thought to correspond to the positive and negative
feedback effects of progesterone (Barraclough et al., 1986).
Tissue preparation and immunocytochemistry. Animals were
deeply anesthetized with tribromoethanol (Aldrich, Milwaukee, WI), and
a 1-2 ml blood sample was taken from the right atrium of the heart
immediately before perfusion for steroid analysis. After a brief rinse
with normal saline (50-100 ml), each rat was perfused transcardially
with ice-cold 4% paraformaldehyde (Electron Microscopy Sciences, Fort
Washington, PA) in 0.1 M borate buffer at pH 9.5 for 20 min. The brains were quickly removed, post-fixed for 2 hr in the same
fixative containing 20% sucrose, and then placed in 20% sucrose in
0.02 M potassium PBS (KPBS) for cryoprotection. Thirty-micrometer-thick frozen sections through the AVPV of each brain were collected, at a frequency of one of four, in chilled 0.02 M KPBS. Free-floating sections were incubated in
anti-GluR1, anti-GluR2/3, GluR4 (raised in rabbit; Chemicon, Temecula,
CA), GluR5/6/7 (mouse monoclonal antibody; PharMingen, San Diego, CA), and anti-NMDAR1 primary antisera (raised in rabbit; Chemicon) at 4°C
with constant agitation for 72 hr. The GluR1, GluR2/3, and GluR4
antibodies were generated from a C-terminus peptide of rat GluR1,
GluR2, and GluR4, respectively. GluR5/6/7 antibody was prepared against
a fusion protein from the N-terminal putative extracellular portion of
GluR5, which belongs to the kainate type of glutamate receptor, and
affinity purified using the immunogen peptide (Wenthold et al., 1992;
Huntley et al., 1993). The anti-GluR1 recognizes GluR1, the
anti-GluR2/3 recognizes GluR2 and GluR3, the anti-GluR4 recognizes
GluR4, and the anti-GluR5/6/7 recognizes GluR5, GluR6, and GluR7 but
not other members of the GluR family. The NMDAR1 antibody was generated
from a synthetic peptide corresponding to the C terminus of rat NMDAR1
subunit and affinity purified using the immunogen peptide. Selective
for splice variants NMDAR1-1a, NMDAR1-1b, NMDAR1-2a, and NMDAR1-2b,
this antibody is specific for NMDAR1 and does not appear to cross-react
with other glutamate receptor subunits (Petralia et al., 1994). The
GluR1, GluR2/3, GluR4, GluR5/6/7, and NMDAR1 antibodies were diluted to
1:4000, 1:2000, 1:1000, 1:2000, and 1:500, respectively, in KPBS that contained 2% normal goat serum (Colorado Serum Co.) and 0.3% Triton X-100 (Bio-Rad, Hercules, CA). After brief rinses in KPBS containing 0.3% Triton X-100, the sections were then incubated in a biotinylated goat anti-rabbit IgG secondary antiserum (Vector Laboratories, Burlingame, CA) at room temperature. The sections were rinsed in KPBS
and stained by using the ABC method (Hsu et al., 1981) with commercial
reagents (Elite kit; Vector Laboratories) at room temperature, and the
incubations in the secondary antiserum and ABC solution were repeated,
followed by several rinses in KPBS. The sections were then
color-reacted with 0.03% diaminobenzidine (Sigma), 2.5% nickel
ammonium sulfate, 0.2% D-glucose, 0.04% ammonium chloride, and 0.001% glucose oxidase (Sigma) in 0.1 M
acetate buffer. All sections were mounted on gelatin-coated microscopic slides, air dried, dehydrated, and coverslipped.
In situ hybridization. Twenty-micrometer-thick frozen
sections (at a frequency of one of four) through the AVPV of
each brain were collected in chilled 0.02 M KPBS that
contained 0.25% paraformaldehyde, pH 7.4, mounted onto
gelatin-subbed, poly-L-lysine-coated microscopic slides,
and processed for in situ hybridization as described
previously (Simmons et al., 1989). To control for procedural artifacts,
all tissue hybridized with each probe was processed together in a single in situ hybridization histochemistry experiment.
After a 30 min proteinase K digestion (10 µg/ml at 37°C; Boehringer Mannheim, Indianapolis, IN) and acetylation (0.0025% acetic anhydride at room temperature), the sections were dehydrated in ascending alcohols and dried under vacuum overnight. T7 polymerase
(Promega, Madison, WI) was used to transcribe 35S-labeled
antisense cRNA probes from a 721 bp PstI fragment of plasmid
pBluescript SK( ) complementary to the 5' coding region of rat
glutamate receptor subunit gene GluR1, from a 428 bp SphI fragment complementary to the 5' coding region of rat glutamate receptor subunit gene GluR2, or from a 723-bp SarI fragment,
which is complementary to the 5' coding region of rat glutamate
receptor subunit gene GluR3 (all three probes were kindly provided by
Dr. S. Heinemann, The Salk Institute, La Jolla, CA). For the
NMDAR1 probe, T3 polymerase (Promega) was used to transcribe a
35S-labeled antisense cRNA probe from an 868 bp cDNA insert
corresponding to nucleotides 699-1567 of the rat NMDAR1 gene sequence
(Moriyoshi et al., 1991) contained in a pBluescript SK()
transcription vector (Urbanski et al., 1994). T3 or T7 polymerase was
also used to transcribe 35S-labeled antisense cRNA probes
from cDNA inserts corresponding to metabotropic glutamate receptor
(mGluR) subtypes: mGluR1 (nucleotides 3541-4282), mGluR2 (nucleotides
2712-3294), mGluR3 (nucleotides 2376-3215), mGluR4 (nucleotides
2910-3704), mGluR5 (nucleotides 1246-1786), and mGluR6 (nucleotides
3669-4418). The mGluR plasmids were generously provided by Dr.
S. Nakanishi (Kyoto University, Kyoto, Japan), and mGluR 5 subclone was
provided by Drs. Y. J. Ma and S. Ojeda (Oregon Regional Primate
Research Center). The radiolabeled cRNA probe was purified by passing
the transcription reaction solution over a Sephadex G-50 Nick column
(Pharmacia, Piscataway, NJ), and four 100 µl fractions were collected
and counted by using a scintillation counter (Packard, Meridian, CT). The leading fraction was heated at 65°C for 5 min with 500 µg/ml yeast tRNA (Sigma) and 50 µM dithiothreitol (DTT)
(Stratagene, La Jolla, CA) in DEPC (Sigma) water and then diluted to an
activity of 5 × 106 with hybridization buffer
containing 50% formamide (Boehringer Mannheim), 0.25 M
sodium chloride, 1× Denhardt's solution (Sigma), and 10% dextran
sulfate (Pharmacia). This hybridization solution was pipetted onto the
sections (80 µl/slide), which were covered with a glass coverslip,
and sealed with DPX (Electron Microscopy Sciences) before incubation
for 20 hr at 58°C. After hybridization, the slides were washed four
times (5 min each) in 4× SSC before RNase digestion (20 µg/ml for 30 min at 37°C; Sigma) and rinsed at room temperature in decreasing
concentrations of SSC that contained 1 mM DTT (2×, 1×,
0.5× for 10 min each) to final stringency of 0.1× SSC at 65°C for
30 min. After dehydration in ascending alcohols, the sections were
exposed to DuPont (Billerica, MA) Cronex x-ray films for 4 and 8 d, together with autoradiographic 14C microscales
(Amersham, Arlington Heights, IL), before being dipped in NBT-2 liquid
emulsion (Eastman Kodak, Rochester, NY). The dipped autoradiograms were
developed 21 d later with Kodak D-19 developer, and the sections
were counterstained with thionin through the emulsion.
Double in situ hybridization. The method used in
this study was a modification of that reported by Springer et al.
(1991) and described in detail previously (Simerly et al., 1996).
Briefly, in vitro transcription of the GluR1 insert was
performed as described above, except that 1 µl of a 2 mM
solution of digoxigenin-labeled UTP (Boehringer Mannheim) was
substituted for the isotope 35S-labeled UTP (DuPont NEN,
Boston, MA). After incubation with T7 polymerase, the reaction mixture
was treated with DNase (Promega) and RNAsin (Promega) and stabilized
with EDTA (Sigma) and salt, and the total volume was adjusted to 100 µl with 20 mM DTT. The cRNA probe was then precipitated
with ethanol, dried, and resuspended in 100 µl of DEPC-treated water.
A total of 150 µl of digoxigenin-labeled cRNA probe was diluted in 1 ml of hybridization solution containing 35S-labeled GluR2
probe as prepared above. Prehybridization, hybridization, and
posthybridization procedures were basically identical to those described above, except that the sections were not dehydrated and dried
after the last 0.1× SSC rinse but were processed for further
localization of digoxigenin-labeled hybrids. Before immunohistochemical detection of digoxigenin-labeled hybrids, the slides were incubated overnight in 2× SSC containing 0.05% Triton X-100 and 2% normal goat
serum at room temperature. The next day, the slides were incubated in a
1:1000 dilution of the anti-digoxigenin-alkaline phosphatase conjugate
(Boehringer Mannheim) for 5 hr at room temperature, rinsed, and then
incubated overnight at room temperature in the chromogen solution, and
the staining reaction was stopped by placing the slides in 10 mM Tris-HCl with EDTA. The sections were further dehydrated
in ethanol (containing SSC and DTT), dried under vacuum for 30 min, and
exposed to DuPont Cronex x-ray film for 4 d before being dipped in
Ilford K5 emulsion (Polysciences, Warrington, PA) for autoradiography
to detect 35S-labeled hybrids. After a 3 week exposure, the
emulsion-coated slides were developed as described above, washed,
dehydrated in ethanol, dried under vacuum for 30 min, coverslipped with
DPX, and evaluated at a high magnification (400×).
Quantification and analysis. The optical density of the
autoradiographic images of GluR1, GluR2, GluR3, and NMDAR1 mRNAs on x-ray films was measured by using a Macintosh-based image analysis system and NIH Image software from National Institutes of Health. Each
film was illuminated with a ChromaPro 45 light source, which provided
even illumination, and the image was obtained with a Dage-MTI (Michigan
City, IN) 70 series video camera equipped with a Newvicon tube. The
optical density of autoradiographic images over the AVPV (4 d exposure)
was measured on film at the same level from each brain. The boundaries
of the AVPV were determined from observation of the corresponding
Nissl-stained sections. A total of eight frames of images were
averaged, and the film densities were integrated over the entire AVPV.
The mean optical density over a large irregularly shaped region over
the third ventricle adjacent to the AVPV that did not contain specific
hybridization signals was also measured on each section and used to
calculate the mean background density, which was subtracted from the
optical density measurement of signals over the AVPV. Although these
mean optical density measurements do not correspond to absolute optical density units, they reflect relative mRNA levels in the AVPV. Commercially available 14C autoradiographic standards were
exposed to each x-ray film along with the experimental material. The
mean optical density of an interactively defined region over each
standard was measured; these measurements confirmed the linearity of
the responsiveness of the film, as well as the consistency of
signal detection across films. The mean optical densities of the
autoradiographic images recorded over the AVPV all fell within the
linear range of the standard values. Ratios between levels of
GluR1/GluR2 mRNA were calculated by dividing the optical density of
GluR1 mRNA by the optical density of GluR2 mRNA measured for adjacent
sections from the same animal. Similarly, GluR3/GluR2 mRNA ratios were
also calculated. A two-way ANOVA was used to test for significant
differences in densities of GluR1-3 or NMDAR1 mRNAs among groups in
each experiment, and a post hoc Fisher's test was
used to identify significant differences between individual groups.
p  0.05 was defined as significant. The
colocalization of GluR1 and GluR2 mRNA was analyzed by counting, at a
magnification of 400×, the number of darkly stained
digoxigenin-labeled GluR1 mRNA-containing neurons, the number of GluR2
mRNA-containing neurons, visualized by clusters of silver grains at a
density of three times greater than that of background, and the number
of double-labeled cells contained within the morphological borders of
the AVPV.
Ultrastructural analysis. To confirm that NMDAR1
immunoreactivity was located in terminals that synapse onto AVPV
neurons, sections were prepared for electron microscopic analysis. Two ovariectomized juvenile female rats that had been treated for 48 hr
with estradiol as described above were deeply anesthetized with
tribromoethanol (Aldrich) and perfused transcardially with 100 ml of
normal saline, followed by 300 ml of ice-cold fixative containing 4%
paraformaldehyde, 15% picric acid, and 0.08% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 20 min. The brains were quickly removed, post-fixed for 2 hr in the same fixative without
glutaraldehyde, and then rinsed in PB. Fifty-micrometer-thick vibratome
sections through the AVPV were collected in PB, transferred for 20 min
in PB containing 10% sucrose for cryoprotection, rapidly frozen by immersion in liquid nitrogen, and thawed at room temperature. After several rinses in PB, they were further incubated for 10 min in
1% sodium borohydride (Sigma) in PB to eliminate unbound aldehydes and
thoroughly washed in PB. The sections were incubated for 72 hr at 4°C
under agitation in a rabbit anti-NMDAR1 antiserum (Chemicon), diluted
to 1:500 in PB containing 2% normal goat serum. After several washes,
sections were transferred to a biotinylated goat anti-rabbit IgG
secondary antiserum (Vector Laboratories) for 2 hr at room temperature,
rinsed in PB, and incubated in ABC solution (Elite kit; Vector
Laboratories) for 2 hr at room temperature. The tissue-bound peroxidase
was visualized by a DAB reaction (10 mg DAB and 5 µl of hydrogen
peroxide in 30 ml PB). After extensive washes, sections were osmicated
in 1% osmium tetroxide in PB for 15 min, rinsed, dehydrated in an
ascending gradient of alcohols and in propylene oxide, and left for 2 hr in propylene oxide/araldite (1:1). The tissue was polymerized in
araldite for 48 hr at 60°C, and ultrathin sections were cut on a
Reichert ultratome and examined under a Jeol-1200 EX (Peabody, MA)
electron microscope.
Hormone assays. Blood samples that were taken immediately
before perfusion were collected in Eppendorf tubes, left standing for
coagulation at room temperature for 2 hr, and stored at 4°C for 24 hr. Serum was separated by centrifugation and stored at 20°C until
assayed for E2 and progesterone by RIA as described previously (Resko
et al., 1975; Hess et al., 1981). All the samples in each experiment
were run in a single assay, with an intra-assay variation of <8%, and
the lower limits of detection were 5 pg/tube in the estrogen assay,
<3.2% intra-assay variation, and 12 pg/tube lower limits of detection
in the progesterone assay.
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RESULTS |
Distribution of glutamate receptors in the AVPV
Each part of the preoptic region contained neurons that
were immunoreactive for ionotropic GluR1-3 receptor subunits; however, each subtype showed a unique distribution. GluR1-immunoreactive cell
bodies and fibers were particularly dense in the AVPV, relative to the
significantly lower levels of staining in surrounding areas (Fig.
1A). Moreover, the
intensity of GluR1 immunostaining in the AVPV was noticeably greater in
fibers than in cell bodies. The density of GluR2/3-immunoreactive
neurons in the AVPV was much lower than that of GluR1-immunoreactive
cells and appeared to be clustered in the medial part of the nucleus
(Fig. 1B). A similar density of GluR2/3 neurons were
localized to the median preoptic and periventricular preoptic nuclei.
In contrast with GluR1 immunoreactivity, GluR2/3 staining in the AVPV
was primarily found in neuronal cell bodies, with only a low density of
labeled fibers present. AVPV neurons do not appear to express
detectable levels of GluR4 immunoreactivity, and only a few
GluR4-immunoreactive fibers were observed within the borders of the
nucleus (Fig. 1C). Furthermore, we found no evidence of
GluR5/6/7 immunoreactivity in the AVPV. However, the majority of AVPV
neurons appear to express NMDAR1 immunoreactivity (Fig.
1D). The AVPV also contains a moderately dense plexus
of NMDAR1-immunoreactive fibers, which have a fine and rather punctate
morphology. Ultrastructural examination of these NMDAR1-immunoreactive
fibers confirmed that staining was contained in axon terminals, which
synapse with dendritic processes and soma of AVPV neurons (Fig.
2A,B);
some of these AVPV neurons were also NMDAR1-immunoreactive. In
situ hybridization was used to determine whether specific mRNAs
corresponding to metabotropic glutamate receptor subunits are expressed
in the AVPV. No mGluR1-6 mRNA-containing neurons were found in the
AVPV, although labeled mGluR2 and mGluR5 neurons were detected
in neighboring regions, such as the median preoptic nucleus.
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Figure 1.
Distribution of GluR1 (A),
GluR2/3 (B), GluR4 (C), and
NMDAR1(D) immunoreactivity in the AVPV of
juvenile female rats. V3, Third ventricle. Scale bar, 50 µm.
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Figure 2.
Subcellular localization of NMDAR1 immunostaining
in the AVPV. At the ultrastructural level, NMDAR1 immunoreactivity is
observed in axonal terminals (asterisks) synapsing on
unlabeled dendritic processes (A, double
arrowheads) or on NMDAR1-immunoreactive dendrites
(B, arrows). Postsynaptic
NMDAR1-immunoreactive elements, such as the dendrite
(d) in A, were frequently observed
receiving an asymmetric synapse (A,
arrows) from unlabeled terminals. Scale bar, 0.5 µm.
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Hormonal regulation of GluR1, GluR2, GluR3, and NMDAR1 gene
expression in the AVPV
Regulation by estrogen
In general, the densities of GluR1-, GluR2-, GluR3-, and NMDAR1
mRNA- containing neurons in the AVPV appear to be higher compared with
the corresponding densities of immunoreactive neurons. This discrepancy
may be attributable to the limited sensitivity of the
immunocytochemical staining or a high turnover rate of the corresponding proteins. Treatment of ovariectomized juvenile female rats with E2 resulted in elevated levels of serum E2 (68.5 ± 8.5 pg/ml; baseline value, 28.5 ± 6.2) within the physiological
range, consistent with previous reports (Andrews et al., 1981).
Forty-eight hours after E2 treatment, levels of GluR1 mRNA
hybridization were increased in the AVPV by over 35% compared with
those of ovariectomized animals treated with control capsules (Figs. 3,
4A,B).
Similarly, estradiol treatment caused a 50% increase in the ratio of
GluR1/GluR2 mRNA (Fig. 3). However, no significant differences (two-way
ANOVA) in levels of GluR2 and GluR3 mRNAs (Fig.
5) or in the ratio of GluR3/GluR2 mRNA
were identified between experimental and control groups. In contrast
with GluR1 mRNA regulation, estradiol appears to suppress NMDAR1 mRNA
levels by ~50% in the AVPV (Figs.
4C,D, 6).
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Figure 3.
Comparison of hybridization signals for GluR1
(A) and GluR1/GluR2 (B)
mRNA ratios in the AVPV of experimental and control groups of juvenile
female rats. The ratio of GluR1/GluR2 mRNA was calculated by dividing
the optical density of GluR1 mRNA by the optical density of GluR2 mRNA
in adjacent sections from the same animal. Bars
represent the mean ± SEM density (minus background) of
autoradiographic signals over the AVPV (A) or
mean ratios (B) for each experimental group.
*p < 0.05, significant differences between
parallel experimental and control groups. E2,
Estradiol; OVX, ovariectomy; P4,
progesterone.
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Figure 4.
Photomicrographs that illustrate the
effects of E2 on expression of GluR1 (A,
B) and NMDAR1 (C, D) mRNA
in the AVPV neurons of juvenile female rats. V3, Third
ventricle. Scale bar, 50 µm.
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Figure 5.
Comparison of hybridization signals for GluR2 and
GluR3 mRNAs in the AVPV of experimental and control groups of juvenile
female rats. Bars represent the mean ± SEM density
(minus background) of autoradiographic signals over the AVPV for each
experimental group. There were no significant differences between
parallel experimental and control groups. E2, Estradiol;
OVX, ovariectomy; P4, progesterone.
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Figure 6.
Hybridization signals measured for
NMDAR1 mRNA level in the AVPV of experimental and control groups of
juvenile female rats. Bars represent the mean ± SEM density (minus background) of autoradiographic signals over the
AVPV for each experimental group. *p < 0.05, significant difference between parallel experimental and control
groups. E2, Estradiol; OVX, ovariectomy;
P4, progesterone.
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Regulation by progesterone
Treatment of E2-primed ovariectomized juvenile female rats with
progesterone resulted in a marked increase in serum progesterone (32.5 ± 3.2 ng/ml; baseline value, 6.7 ± 0.9) by 3 hr after
injection. Progesterone levels returned to baseline by 24 hr after
injection. GluR1 mRNA levels in the AVPV were increased by over
26% in rats killed 3 hr after progesterone injection and returned to
baseline by 24 hr (Fig. 3). However, progesterone did not appear to
cause significant changes in levels of GluR2 or GluR3 mRNAs in the AVPV (Fig. 5). Surprisingly, short-term progesterone treatment did not
significantly alter the ratio of GluR1/GluR2 mRNA (Fig. 3) nor the
GluR3/GluR2 mRNA ratio. Longer exposure to progesterone did cause a
decrease in the GluR1/GluR2 mRNA ratio by 24 hr (Fig. 3). Neither
short-term nor 24 hr exposure to progesterone affected overall levels
of NMDAR1 mRNA in the AVPV (Fig. 6).
Coexpression of GluR1 and GluR2 mRNAs in the AVPV
Double in situ hybridization was used to examine
coexpression of GluR1 and GluR2 in the AVPV. The degree of
colocalization suggests that nearly all of the GluR2 mRNA-containing
neurons also express GluR1 mRNA (Fig. 7).
However, only 85% of the GluR1 mRNA-containing neurons were found to
contain detectable levels of GluR2 mRNA. Although E2 treatment clearly
induces expression of GluR1 mRNA, it did not appear to influence the
percentage of colocalization or alter the number of GluR1
mRNA-containing neurons in the AVPV.
View larger version (121K):
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Figure 7.
Combined bright-field-epi-illumination
photomicrograph of GluR1 mRNA-containing neurons (dark cells) in the
AVPV, visualized with a digoxigenin-labeled cRNA probe, double labeled
for GluR2 mRNA (bright silver grains) by using a
35S-labeled cRNA probe. Arrows indicate
double-labeled GluR1 and GluR2 mRNA-containing neurons.
Arrowheads indicate single-labeled GluR1 mRNA-containing
neurons. Scale bar, 20 µm.
|
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DISCUSSION |
The results of our analysis of glutamate receptor expression in
the AVPV indicate that a somewhat restricted complement of receptor
subtypes is expressed by these neurons. No evidence for expression of
metabotropic receptors (mGluR1-6) was obtained. AMPA and NMDA
receptors appear to be expressed in the greatest abundance with GluR1
mRNAs being present in the highest levels. The AVPV occupies an
important position in neuroendocrine neural circuits (Simerly, 1997)
and may represent a particularly important site for interactions
between sex steroid hormones and EAAs because it contains a high
density of AMPA and NMDA receptors and contains an abundance of neurons
that express ovarian steroid hormone receptors. Thus, the hormonal
regulation of glutamate receptor expression demonstrated in this study
may contribute to the positive feedback of ovarian steroids on
gonadotropin secretion. Previous studies have shown that both sex
steroids and glutamate receptors are essential for the LH surge (for
review, see Brann, 1995). However, few of the GnRH neurons that project
to the median eminence appear to express glutamate receptors (Abbud and
Smith, 1995; Urbanski et al., 1996), suggesting that an indirect
mechanism may be involved in the induction of an LH surge by excitatory
amino acids.
Both EAAs and E2 can promote gonadotropin secretion through the
activation of GnRH neurons in vivo (for review, see Brann, 1995; Urbanski et al., 1996). These effects may be attributable to
interactions between sex steroids and EAA-containing neural pathways
because pharmacological blockade of either steroid hormones or EAA
receptors abolishes their effects on gonadotropin secretion (Estienne
et al., 1990; Brann and Mahesh, 1991a,b, 1992; Reyes et al., 1991;
Brann et al., 1993; Lee et al., 1993; Luderer et al., 1993; Brann,
1995). Hormonal signals may influence glutamatergic neurotransmission
in the brain by regulating the expression of different ionotropic
glutamate receptor subunits (Diano et al., 1997), resulting in the
elaboration of receptors with different properties. Glutamate receptor
subunits are expressed differentially during normal development
(Pellegrini-Giampietro et al., 1991; Bahn et al., 1994; Sheng et al.,
1994) and in response to environmental changes (Pellegrini-Giampietro
et al., 1992; Pollard et al., 1993; Prince et al., 1995; Fitzgerald et
al., 1996). Our results demonstrate that E2 specifically promotes the
expression of GluR1 AMPA type subunits in the AVPV of juvenile rats but
leaves levels of GluR2 and GluR3 unchanged. This result is consistent
with that of earlier studies, which found that the expression of
non-NMDA receptors (Weiland, 1992), and specifically the GluR1 subunit,
is increased by treatment with E2 plus progesterone (Brann and Mahesh,
1994) or by estrogen treatment alone (Weiland, 1992; Ulibari and
Akesson, 1993), and AMPA receptor binding levels in the hypothalamus
were increased at the time of puberty in the female rat (Zamorano et al., 1998). Although these previous studies did not address hormonal regulation of GluR2 expression, changes in levels of GluR2 mRNA appear
to occur elsewhere in the developing brain (Pellegrini-Giampietro et
al., 1991).
The observed regulation of glutamate receptor expression by sex steroid
hormones appears to be specific for the AVPV, because the same pattern
does not occur in the lateral septal nucleus in which estradiol appears
to upregulate levels of NMDAR1 mRNA but leaves the levels of GluR1 mRNA
unchanged (Varoqueaux et al., 1997).
Estrogen may potentiate glutamatergic neurotransmission in the AVPV by
its induction of GluR1 expression and lack of effect on GluR2 mRNA,
which leads to an increase in the GluR1/GluR2 ratio. Ionotropic
glutamate receptors are thought to have a pentameric structure (for
review, see Hollmann and Heinemann, 1994), and a single neuron can
express different combinations of AMPA receptor subunits (Lambolez et
al., 1992). AMPA subunits may form homomeric ion channels or combine
with other subtypes to constitute heteromeric receptors, which exhibit
a wide range of functional diversity that is dependent on the
complement of subunits present. A close correlation between
steady-state levels of mRNAs for distinct GluR subunits and the
functional properties of glutamate-activated channels has been
demonstrated for other neurons (Lambolez et al., 1992; Geiger et al.,
1995; Chew et al., 1997), and the high percentage of AVPV neurons that
coexpress GluR1 and GluR2 subunits suggests that the majority of AMPA
receptors in the AVPV may be heteromeric. In a heteromeric AMPA
receptor channel, GluR2 subunits dominate the rectification properties
and decrease permeability to calcium (Hollmann and Heinemann, 1994;
Geiger et al., 1995). However, other AMPA subunits appear to enhance
the Ca2+ permeability of individual channels. Thus,
a cellular consequence of E2 positive feedback may be an increase in
the calcium permeability of the ionotropic channels in AVPV neurons
brought about by upregulation of the GluR1 subunits and the subsequent
increase in the ratio of GluR1/GluR2. The increase in the GluR1/GluR2
ratio effected by estrogen contrasts with the decrease in this ratio
caused by progesterone 24 hr after treatment. Although progesterone
caused a significant increase in levels of GluR1 mRNA 3 hr after
treatment, there was no significant change in the GluR1/GluR2 mRNA
ratio, perhaps because of a small, statistically insignificant increase in GluR2 mRNA. However, the ratio of GluR1/GluR2 mRNA was significantly decreased by 24 hr after treatment with progesterone. These findings indicate that E2 and progesterone may have opposing actions on the
ratio of GluR1/GluR2 gene expression, which therefore suggests they may
exert opposing effects on glutamatergic neurotransmission in the AVPV.
Whereas estrogen potentiates neuronal firing in the AVPV, progesterone
may suppress the activity of these neurons, which may contribute to the
termination of the positive feedback after the LH surge is generated.
That E2 treatment suppresses NMDAR1 mRNA in the AVPV is a surprising
observation because E2, progesterone, nor testosterone affected
NMDA receptor binding or mRNA levels in adult male and female rats or
in juvenile female rats. This discrepancy may be attributable to
differences in age, sex, or hormonal treatment models but is more
likely to be because of the fact that NMDA receptor expression in the
AVPV was not specifically addressed in the earlier studies.
Furthermore, the presence of NMDAR1 terminals in the AVPV, as
demonstrated in the present work, may render the downregulation of NMDA
receptor expression in AVPV neurons less remarkable in binding studies,
which cannot distinguish between presynaptic and postsynaptic receptors.
The NMDAR1 subunit is an essential part of NMDA receptors. Therefore, a
decrease in its expression may reflect an overall decrease in levels of
NMDA receptors in the AVPV. The results of previous studies indicate
that the selective activation of AMPA receptors leads to a moderate,
nontoxic elevation of intracellular free calcium (50-100%), whereas
activation of NMDA receptors results in a 300-400% increase in
intracellular free calcium, which can be neurotoxic (Cheng and Mattson,
1992). Estrogen has been shown to protect neurons against excitotoxic
damage (Behl et al., 1997; Simpkins et al., 1997), and downregulation
of NMDA receptors may contribute to its neuroprotective action.
Therefore, estrogen may enhance AMPA receptor-mediated
neurotransmission in the AVPV, while at the same time preventing NMDA
receptor-mediated neurotoxicity, which may be important during positive
feedback before the LH surge when levels of glutamate in the preoptic
area are elevated (Jarry et al., 1992; Ping et al., 1994b).
NMDA is also thought to act presynaptically, and the presence of NMDAR1
subunit in presynaptic neuronal elements has been documented (Liu et
al., 1994; Farb et al., 1995; Aicher et al., 1997; Gracy et al., 1997;
Van Bockstaele and Colago, 1997). NMDAR1 immunoreactivity is clearly
present in presynaptic terminals in the AVPV, which terminate on both
cell soma and dendrites. These subunits may form autoreceptors, as
demonstrated in the spinal cord (Liu et al., 1994), and activation of
these receptors may trigger a release of glutamate from these
terminals. Although other studies report that progesterone increases
glutamate release in the preoptic area, the precise role of presynaptic
NMDA receptors in the AVPV remains unknown.
The functional significance of the differential regulation of AMPA and
NMDAR1 glutamate receptor subunits in the AVPV for the neuroendocrine
control of reproduction remains unexplored. It seems unlikely that GnRH
neurons are unresponsive to glutamate, but clear evidence for direct
activation is lacking. Furthermore, we did not evaluate the expression
of either KA1 or KA2 subtype kainate receptor in the AVPV, and
approximately one-third of GnRH neurons appear to express KA2 receptors
(Eyigor and Jennes, 1997). Nevertheless, as a nodal point in neural
pathways mediating hormonal control of gonadotropin secretion, the AVPV
is a likely site for interactions between ovarian steroids and
glutamatergic neurotransmission affecting the preovulatory LH surge.
Moreover, the AVPV receives inputs from other forebrain regions that
convey a variety of sensory modalities known to modulate gonadotropin
secretion, including olfaction and visceral sensory information (for
review, see Simerly, 1997). Thus, by altering the sensitivity of AVPV
neurons to glutamatergic activation, ovarian steroids may regulate the
relative impact of this sensory information on GnRH neurons, thereby
coordinating reproductive responses with environmental constraints.
| |
FOOTNOTES |
Received Sept. 22, 1998; revised Dec. 1, 1998; accepted Feb. 2, 1999.
This work was supported by National Institutes of Health Grants
NS26723, RR00163, and HD18185. We thank Dr. D. Hess for steroid hormone
assays, Drs. S. Heinemann and S. Nakanishi for providing plasmids, Dr.
C. Meshul for the use of electron microscope, J. H. Yu, J. C. Zee, M. A. Kirigiti, and M. A. Ibanez for expert technical
assistance, and C. Houser for preparation of this manuscript.
Correspondence should be addressed to G. B. Gu, Division of
Neuroscience, Oregon Regional Primate Research Center, 505 N.W. 185th
Avenue, Beaverton, OR 97006.
| |
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