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Department of Anatomy and Neurobiology University of Kentucky College of Medicine, Lexington, Kentucky 40536
Address all correspondence and requests for reprints to: Lothar Jennes, Ph.D., Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, 428 Health Science Research Building, Lexington, Kentucky 40502. E-mail: ljenn0{at}pop.uky.edu
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To determine whether administration of kainate is sufficient to induce c-Fos in GnRH neurons, steroid-primed animals received iv injections of subseizure-inducing amounts of kainic acid and were processed for immunohistochemistry and in situ hybridization. The results show that kainic acid causes a significant increase in circulating LH; however, it does not induce c-Fos in GnRH neurons, nor does it cause an increase in GnRH mRNA.
Together, the results suggest that a large subset of GnRH neurons expresses KA2 as well as GluR5 receptor subunits, which would allow the formation of functional glutamate receptor channels, and that this subset of GnRH neurons is activated during the steroid-induced LH surge.
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(ER), and they respond to estradiol with a
transient expression of the transcription factor c-fos (5)
as well as increased TH messenger RNA (mRNA) levels (6). Similarly,
glutamatergic neurons in the hypothalamus contain ER, and
administration of estradiol causes an increase in glutamate content
(7). As glutamatergic synaptic input to GnRH neurons has been shown in
the monkey (8), and noradrenergic synapses occur in the rodent (9), it
can be suggested that released neurotransmitter binds to and activates
specific membrane receptors located on the plasma membrane of GnRH
neurons. For instance, 1B-adrenergic receptor
protein has been identified on most GnRH neurons (10), and KA2 subunits
of ionotropic glutamate receptors have been described in about 40–50%
of the GnRH neurons (11). The distribution of the KA2 receptor subunit
expressing GnRH neurons is reminiscent of the distribution of a
subpopulation of GnRH neurons that synthesizes the transcription factor
c-Fos during the preovulatory surge. These c-Fos-positive GnRH neurons
are located preferentially next to the organum vasculosum of the lamina
terminalis (OVLT), and they account for about 40–50% of all GnRH
neurons (12, 13). Although the exact role of c-Fos in GnRH neurons
remains to be clarified, the transient expression of the transcription
factor has been a useful indicator of cell activation, and it has been
proposed that this c-Fos-positive subset of GnRH neurons initiates or
drives the GnRH-mediated LH surge (13). Thus, the aims of the present
study were to determine whether the subset of GnRH neurons that
expresses KA2 receptor subunits also expresses c-Fos during the surge,
and if the administration of kainic acid is sufficient to induce c-Fos
protein or to stimulate GnRH synthesis in GnRH neurons. Lastly, as KA2
receptor subunits do not assemble into functional homomeric receptor
channels (14, 15), a partner kainate-preferring glutamate receptor
subunit needed to be identified. |
Materials and Methods |
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Immunohistochemistry
Animals were deeply anesthetized with
ketamine-acepromazine maleate (50 and 5 mg/kg BW, respectively), and
blood was collected by heart puncture for RIA of LH. Animals were then
perfusion fixed with 4% paraformaldehyde and 7.5% saturated picric
acid in 0.1 M phosphate buffer (pH 7.4; 400 ml/animal).
Brains were removed and postfixed overnight in above fixative. Serial
30-µm thick coronal vibratome sections were collected in five
series from the region between the medial septum and the
suprachiasmatic nucleus and kept in cryoprotectant at -20 C until use.
At least five sections per animal/experiment were analyzed.
Double immunohistochemistry
Sections were washed in Tris-HCl buffer (0.05 M; pH
7.6) to remove the cryoprotectant and incubated in blocking buffer
(10% normal horse serum, 0.2% Triton X-100, and 0.1% sodium azide in
Tris-HCl buffer) for 1 h. Sections were then incubated in rabbit
anti-c-Fos antibody (AP-5, Oncogene, Cambridge, MA) at a dilution of
1:30,000 in blocking buffer overnight at room temperature followed by
exposure for 1 h to biotin-conjugated donkey antirabbit IgG
(1:500; Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA). Sections were processed with the ABC Elite Standard kit
(Vector Laboratories, Inc., Burlington, CA) according to
the instructions of the manufacturer and stained with filtered (25 mg;
Millipore Corp., Bedford, MA) diaminobenzidine and nickel
ammonium sulfate (2 g) in the presence of 2 µl
H2O2 (30%)/100 ml sodium
acetate buffer (0.1 M; pH 6.5). The stained sections were
then washed extensively in Tris-HCl buffer and blocking buffer and
incubated overnight in monoclonal mouse anti-GnRH IgG containing tissue
culture supernatant (1:100; AB 4H10, Jennes). Sections were washed in
Tris-HCl buffer and exposed to biotinylated donkey antimouse IgG and
avidin-biotin complex, as described above, and stained with
diaminobenzidine (50 mg and 5 µl
H2O2/100 ml Tris-HCl). GnRH
immunoreactivity was identified as a brown precipitate in the
cytoplasm, and c-Fos immunoreactivity appeared as a black precipitate
over cell nuclei.
Triple and quadruple immunohistochemistry
The staining of c-Fos protein was conducted as described above.
The stained sections were thoroughly washed in Tris-HCl and blocking
buffer and incubated in a mixture of rabbit anti-KA2 (R52–4, Jennes
and Eyigor, 1:2000) and guinea pig anti-GnRH (GP8–4, Jennes, 1:1000)
in blocking buffer for 72 h at 4 C. For quadruple
immunohistochemistry, mouse antiglutamate receptor 5/6/7 (GluR5/6/7,
1:400, PharMingen, San Diego, CA) was added to this
mixture. After two washes in Tris-HCl buffer of 15 min each, sections
were incubated in a mixture of affinity-purified and cross-absorbed
second antibodies that were labeled with different fluorescent markers
(Jackson ImmunoResearch Laboratories, Inc.). For triple
labeling, Texas Red-labeled donkey antirabbit (1:100) was coincubated
with FITC-labeled donkey antiguinea pig (1:100), and for quadruple
labeling, a Cy-5-labeled second antibody was added. Sections were
washed in Tris-HCl buffer, mounted onto glass slides, dried, and
coverslipped with ProLong antifade mounting medium (Molecular Probes, Inc., Eugene, OR) 1 h before the analysis. Dual and
triple labeled sections were analyzed with an Olympus Corp. BH-2 microscope (New Hyde Park, NY) equipped with the
appropriate excitation-barrier filter combinations, whereas quadruple
labeled sections were observe with a Leica TCS NT confocal microscope
(Leica, Rockleigh, NJ).
Control experiments included absorption of the primary antibodies with 50 µg antigen/ml dilute antiserum, omission of primary antisera, as well as extensive tests of cross-reactivity of the second antibodies. All control experiments resulted in the absence of staining.
In situ hybridization
Deeply anesthetized animals were decapitated, trunk blood was
collected for RIA determinations of LH levels, and brains were removed
and frozen on dry ice. Twelve-micron thick sections were cut on a
cryostat and collected on positively charged slides that were kept in
air-sealed boxes at -80 C until use.
Preparation of complementary RNA (cRNA) probes
Complementary DNA (cDNA) clones complementary to glutamate
receptor subunits GluR5, GluR6, and GluR7 were provided by Dr. Boulter,
Molecular Neurobiology Laboratory at The Salk Institute (La Jolla, CA),
whereas the GnRH cDNA clone was a gift from Dr. Adelman, Vollum
Institute (Portland, OR). Plasmids containing the cDNA clone
complementary to rat GluR5 were linearized with NotI,
whereas the other plasmids were linearized with EcoRI
(GluR6), BamHI (GluR7), or HincII (GnRH) and
transcribed in vitro in the presence of
[35S]UTP and T7 RNA polymerase for antisense
probes. The characteristics of these clones have been described
previously: GluR5, EMBL/GenBank accession no. M83560 (16); GluR6,
accession no. Z11548 (17); GluR7, accession no. M83552 (18); and GnRH,
accession no. M31670 (19).
For dual in situ hybridization experiments, linearized GnRH cDNA was transcribed in vitro in the presence of digoxigenin-11-UTP (Roche Molecular Biochemicals, Mannheim, Germany) with T7 polymerase, resulting in a 275-base long cRNA probe.
Pretreatment, hybridization, and posttreatment
Slides were equilibrated to room temperature and fixed for 15
min in 4% paraformaldehyde, followed by two 5-min rinses in PBS (0.1
M), two 5-min rinses in PBS containing 10 mM
glycine, and two 5-min PBS rinses. The sections were then incubated for
10 min in triethanolamine (0.1 M; pH 8.0) and 0.25% acetic
anhydride, dehydrated in ascending ethanol concentrations, and
air-dried. Sections were hybridized overnight at 60 C with 1.5 x
106 cpm 35S-labeled
kainate-preferring subunit cRNA probe/60 µl·slide and 2 µl
digoxigenin-labeled GnRH cRNA probe in hybridization buffer containing
salmon sperm DNA (100 µg/ml), yeast total RNA (250 µg/ml), yeast
transfer RNA (250 µg/ml), Tris-HCl (pH 7.4; 20 mM), EDTA
(1 mM), NaCl (300 mM), deionized formamide
(50%, vol/vol), dextran sulfate (10%, vol/vol), Denhardt’s solution
(1 x), dithiothreitol (100 mM), SDS (0.1%, wt/vol), and
sodium thiosulfate (0.1%, wt/vol). For single in situ
hybridization, saturating amounts of 35S-labeled
GnRH cRNA probe were used. Sections were then rinsed in 2 x SSC
(standard saline citrate) and treated with ribonuclease (10 mg/100 ml)
for 30 min at 45 C to reduce the nonspecific binding. The sections were
then washed in 0.2 x SSC and kept for 1 h at 60 C in
0.1 x SSC, followed by brief rinses in the same stringency wash
at room temperature and 70% and 95% ethanol concentrations and
air-dried. For dual in situ hybridization, sections were
then incubated in 2% lamb serum in buffer A (100
mM Tris-HCl, pH 7.5, and 150
mM NaCl) with 0.05% Triton X-100 for 2–3 h
followed by an overnight incubation in buffer A containing sheep
antidigoxigenin Fab conjugated to alkaline phosphatase (1:1,000;
Roche Molecular Biochemicals), 1% normal lamb serum, and
0.3% Triton X-100. After washes in buffer A and buffer B (100
mM Tris-HCl, pH 9.5; 100 mM
NaCl; and 50 mM MgCl2),
sections were stained in the dark for 1–3 h in chromogen solution (45
µl nitro blue tetrazolium-chloride, 35 µl
5-bromo-4-chloro-3-indolyl phosphate, and 10 mg levamisole/10 ml buffer
B). The reaction was stopped in buffer B, followed by rapid rinses in
70% and 95% ethanol. Air-dried sections were coated with 3%
parlodion in isoamyl acetate and dried again. The slides were then
dipped in Kodak photographic emulsion NTB2 (Eastman Kodak Co., Rochester, NY), diluted 1:1 with water, kept at 4 C,
in a light- and humidity-free environment, and exposed for 72 h
for single labeling with [35S]GnRH and for 3–8
weeks for dual in situ hybridization. After developing
(Kodak Developer D19) and fixing (Kodak Rapid
Fixer), sections were analyzed with an Olympus Corp. BH-2
microscope. Specificity controls included incubation with
35S-labeled sense probe, pretreatment with
ribonuclease, and coincubation with a 100-fold excess of unlabeled
antisense probe. Control experiments resulted in the absence of
specific labeling.
Image analysis
Each GnRH-immunoreactive neuron was examined for the
presence of KA2 protein in cytoplasm and, when appropriate, for c-Fos
immunoreactivity.
Image analysis of the slide autoradiograms was based on measurements of the areas occupied by silver grains over labeled neurons using NIH Image software. Images were captured, and the gray level threshold was adjusted to allow measurements of pixels corresponding to silver grains. The same threshold setting was maintained for the duration of the analysis. For sampling, individual cells were outlined under x40 oil objective on the basis of grain distribution, and the measurements were taken by subtracting the background.
For dual in situ hybridization experiments, slides were analyzed for the coexistence of silver grains with the blue-brown immunostaining reaction product. GnRH neurons were considered double labeled if the number of silver grains over the neurons was at least 3 times higher than background.
Data analysis
Data are expressed as the mean ± SEM for each
animal, and then using the individual animal values, a grand mean
± SEM were determined for each group. Data then were
subjected to two-way ANOVA, followed by Student’s t test.
Differences were considered statistically significant at
P < 0.05.
RIA for LH
Plasma LH was measured by a standard double antibody RIA, using
the reagents provided by the National Hormone and Pituitary Program,
NIDDK. Purified rat LH was iodinated with Iodogen (Pierce Chemical Co., Rockford, IL) and separated from free
125I by passage over a Bio-Gel P-60 column
(Bio-Rad Laboratories, Inc., Hercules, CA).
Experiments
Exp 1: Do KA2 subunit expressing GnRH neurons also express c-Fos
during the steroid-induced LH surge? Our previous studies (11, 20)
showed that the KA2 receptor-expressing GnRH neurons are localized in a
distribution pattern similar to that of the subgroup of c-Fos-positive
GnRH neurons (12, 13). To assess whether the two groups of GnRH neurons
are identical or overlap, triple labeling immunohistochemistry was
employed to identify GnRH, KA2 subunit, and c-Fos. Fifty-day-old female
rats were ovariectomized 12 days before sc implantation at 0900 h
of a SILASTIC brand capsule (20 mm long; id, 1.57 mm; od, 3.18 mm;
Dow Corning Corp., Midland, MI) containing estradiol (180
µg/ml in sesame oil). Two days later, the animals received one sc
injection of progesterone (50 mg/kg BW in sesame oil) at 0900 h.
Five groups of animals (n = 4–7) were perfusion fixed as
described above at 1000, 1200, 1400, 1600, and 2000 h on the day
of the progesterone injection. Ovariectomized control animals received
identical sc capsules filled with sesame oil and 2 days later a sc
injection of sesame oil.
After immunohistochemical staining for GnRH, c-Fos, and KA2 receptor
subunit, sections were analyzed with an Olympus Corp. BH-2
microscope with brightfield and fluorescent illumination.
Exp 2: Which kainate-preferring glutamate receptor subunits are
coexpressed with KA2 subunits in GnRH neurons?
KA2 subunits need to assemble with other
kainate-preferring glutamate receptor subunits to form a functional
heteromeric ion channel (14). We used dual in situ
hybridization and triple and quadruple immunohistochemistry to identify
a possible expression of GluR5, GluR6, or GluR7 subunits in GnRH
neurons. Intact adult female rats were killed in the morning and
processed for dual in situ hybridization for GnRH mRNA and
one of the above kainate-preferring subunit mRNAs. To identify the
subunit proteins and a possible relationship to c-Fos-expressing GnRH
neurons, tissues of animals treated as described in Exp 1 were used,
and immunohistochemical procedures were applied to identify
simultaneously GnRH, KA2, Glur5/6/7, and c-Fos as described above.
Exp 3: Does activation of kainate-preferring glutamate receptors by kainic acid cause an increase in GnRH mRNA levels and/or induction of c-Fos synthesis?. Previous studies have shown that administration of kainic acid to estradiol-treated animals induces an increase in GnRH-mediated LH release (3). To determine whether activation of kainate receptors in GnRH neurons can induce c-Fos synthesis and increase GnRH mRNA levels, in situ hybridization for GnRH mRNA and dual immunohistochemistry for c-Fos and GnRH were applied. Two groups of animals (10 animals/group) were ovariectomized for 2 weeks and treated with estradiol as described in Exp 1. The next day, animals were fitted with an atrial catheter through the right jugular vein. At 0900 h on the following day, animals in both groups received a sc injection of progesterone as in Exp 1, and between 1000–1100 h, group 1 received kainic acid (2.5 mg/kg in saline, iv) and group 2 received saline iv. This time point was chosen because estradiol-induced changes in LH release or c-Fos expression in GnRH neurons have not yet occurred. Blood samples (0.2 ml; replaced with 0.2 ml saline) were collected 10 min before and 10 min after the injections for LH RIA. Thirty minutes after kainate or saline injections, animals were decapitated and processed for in situ hybridization with 35S-labeled GnRH cRNA probe (5 animals/group) or were perfusion fixed 2 h after the injections and processed for immunohistochemistry (5 animals/group) as described above.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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). Most of the
c-Fos-positive GnRH neurons were located around the OVLT, caudal medial
septum, and diagonal band of Broca.
Immunohistochemical dual stainings for GnRH and KA2 receptor subunit
protein showed that about 50% of all GnRH neurons contain this kainate
receptor subunit and that this percentage did not change throughout the
day of the estrogen-progesterone-induced LH surge (Fig. 1C). Subsequent
triple stainings for GnRH, c-Fos, and KA2 subunit revealed that at
1400 h, about 50% of the c-Fos-positive GnRH neurons also
contained KA2 subunit protein; this percentage remained high at about
65% at 1600 h and 50% at 2000 h (Fig. 1D). If the data are
expressed as the percentage of total GnRH neurons that contained both
c-Fos and KA2 subunit, then about 30% of all GnRH neurons contained
both c-Fos and KA2 receptor subunit protein at 1400 h; this
percentage significantly increased to about 45% at 1600 h
(P < 0.05) and had declined to about 35% by 2000
h (Fig. 1E). The data are summarized in Table 1, the locations of c-Fos containing GnRH
neurons with and without KA2 receptor subunit protein from a
representative animal are detailed in Fig. 2, and an example of a triple labeled
GnRH neuron containing c-Fos and KA2 subunit protein is shown in Fig. 3, A–D.
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, E–H). As Exp 1 showed that most of the
c-Fos-positive neurons also express KA2 subunit protein,
immunohistochemical quadruple labeling procedures were applied to
determine whether these c-Fos- and KA2-positive GnRH neurons also
contained GluR5/6/7 receptor subunits. The results showed that the GnRH
neurons that contained c-Fos and KA2 subunit protein were also
immunoreactive to GluR5/6/7 antibody (Fig. 3, I–L). As the GluR5/6/7
antibody does not distinguish among the three individual subunit
proteins, dual in situ hybridization for GnRH and GluR5,
GluR6, or GluR7 mRNAs was employed. The results revealed that only
GluR5 mRNA was detected in GnRH neurons (Fig. 4), whereas GluR6 and GluR7 mRNAs were
apparently absent in GnRH neurons (data not shown). About 50%
(50.51 ± 4.36) of GnRH neurons expressed GluR5 mRNA.
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). However, analyses of
autoradiograms generated after in situ hybridization
with 35S-labeled GnRH cRNA probe indicated
that kainic acid treatment did not cause an increase in GnRH mRNA
content 30 min after administration of the drug (Fig. 6). This time interval was chosen based
on data reported by Petersen et al. (21) showing that the
effects of another glutamate agonist,
N-methyl-D-aspartate (NMDA), on GnRH mRNA
content were observed between 15 and 60 min. Similarly, iv
administration of kainic acid did not induce the expression of c-Fos in
GnRH neurons 2 h after the beginning of treatment (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-adrenergic receptor antagonists (26, 27)
prevents the GnRH-mediated LH surge in the presence of gonadal
steroids, which suggests that proper activation of glutamate and/or
adrenergic receptors is required for adequate GnRH release. Recently,
we have shown that KA2 receptors (11) and
1B-adrenergic receptors (10) are expressed by
GnRH neurons, which further underlines the presence of neuronal
projections that regulate the GnRH neurons. However, the functional
significance of the kainate receptors was not clear at that time, as
homomeric assembly of KA2 receptor subunits does not generate active
channels (14), albeit their high affinity for
[3H]kainate (15). Our finding that the GnRH
neurons that express KA2 receptor subunits also contain GluR5 subunit
mRNA and protein suggests that an assembly of KA2-GluR5 subunits is
possible, which would generate functional ion channels that would
exhibit a high affinity for kainate, a prominent rapid current
component, and rapid desensitization (28). The precise role of the
functional kainate receptors in the generation of the LH surge is not
clear, as activation of the kainate receptors by peripheral injections
of agonist causes only a modest increase in GnRH-mediated LH release
(3, 29), whereas GnRH mRNA levels remain unchanged (present study).
These data obtained from female animals are somewhat different from the
recent report by Gore et al. (30), who measured a 1-fold
rise in cytoplasmic GnRH mRNA in male rats after peripheral
administration of kainic acid. It is not clear at present whether the
different results are due to a sexual dimorphism in the response to
kainic acid or to different sensitivities of in situ
hybridization and solution hybridization. Glutamate release in the preoptic area is increased before and during the surge (31), and blockade of glutamate receptors with antagonists prevents the surge (25, 32, 33). These data suggest that activation of glutamate receptors is required for an appropriate surge, but it is not sufficient to induce massive GnRH release. It appears, therefore, that glutamate is probably not the neurotransmitter that is the driving force for the activation of GnRH neuronal activity during the surge but, instead, may be important in setting excitability thresholds of GnRH neurons or modifying the responsiveness to other neurotransmitters.
Our finding that KA2 receptor protein is preferentially expressed in those GnRH neurons that synthesize c-Fos during the surge is intriguing and indicates that this subset of GnRH neurons is functionally different from the other GnRH neurons. The transient presence of c-Fos in neurons has been an useful indicator to distinguish activated from nonactivated cells in many neuroendocrine systems (34), and in the case of the GnRH system, a parallelism between the number of c-Fos-positive GnRH neurons and the magnitude of GnRH-stimulated LH release has been proposed (35). This view is supported by our finding as well as the data reported by Attardi et al. (36) that the percentage of c-Fos-positive GnRH neurons is higher during the steroid-induced surge than during a physiological proestrous surge. The exact role of c-fos in GnRH neurons is not known, but the induction of the c-fos gene does not appear to be required for the generation of a preovulatory surge, as c-fos-deficient mice can reproduce (37). On the other hand, Wang et al. (38) found that c-Fos-expressing GnRH neurons contain more GnRH mRNA than c-Fos-negative GnRH neurons. As the GnRH promoter contains an activating protein-1 site to which a c-Fos heterodimer can bind (19), it is possible that c-Fos expression is related to replenishing GnRH peptide in these activated neurons.
c-Fos expression can be induced in neurons by many different stimuli,
such as growth factors, membrane depolarization, or neurotransmitters
(39). In particular, glutamate agonists have been shown to induce c-Fos
synthesis in several seizure models, although it is not fully
understood at present whether these effects are mediated solely by
activation of the relevant glutamate receptors on the
c-fos-expressing target cells or if an agonist-induced
reduction of an inhibitory 
-aminobutyric acid (GABA) tone
contributes to the induction of c-Fos synthesis (40). In these models,
kainate is the most potent glutamate agonist inducing c-Fos synthesis
(40), and based on our finding that most c-Fos-positive GnRH neurons
also contain KA2 receptors, it can be speculated that activation of
these receptors is responsible for the induction of c-Fos synthesis.
However, as peripheral administration of subseizure levels of kainic
acid fails to induce c-Fos in GnRH neurons, but not in hippocampal
neurons, it is clear that other factors are required to induce c-Fos
synthesis in GnRH neurons. It is possible that other neurotransmitters,
such as, endogenous GABA, for which the GnRH neurons have receptors
(41), prevent c-Fos expression in GnRH neurons during all stages of the
estrous cycle, except during the rising phase of the LH surge, when
GABA release is reduced. To date, all attempts have failed to induce
c-Fos in GnRH neurons by pharmacological treatments with NMDA
(42), kainic acid (this report), adrenergic receptor agonists, GABA
antagonists, or combinations of these (Eyigor, O., and L. Jennes,
unpublished observations), and the only treatment that has been
successful is the administration of gonadal steroids. Based upon the
finding that treatments with pentobarbital or the glutamate antagonist
MK 801 (42) prevent expression of c-Fos in GnRH neurons after treatment
with gonadal steroids, it is apparent that the c-Fos-inducing effects
of the steroids are mediated by activation/inhibition of
neurotransmitter systems that innervate GnRH neurons and not by direct
actions of the steroids on GnRH neurons. It is likely that a precisely
timed activation of glutamate and/or adrenergic receptors and
inhibition of GABA release are required to induce c-Fos synthesis in
GnRH neurons and to induce a robust GnRH-mediated LH surge.
In conclusion, the data of this study suggest that a large subset of GnRH neurons expresses KA2 as well as GluR5 receptor subunits, which would allow the assembly of functional glutamate receptor channels, and that this subset of GnRH neurons is activated during the steroid-induced LH surge, as indicated by the transient synthesis of c-Fos.
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Acknowledgments |
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Footnotes |
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Received July 21, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1B adrenergic receptor protein in gonadotropin
releasing hormone neurones of the female rat. J Neuroendocrinol 10:687–692[CrossRef][Medline]
-immunoreactivity in gonadotrophin-releasing
hormone-expressing neurones. J Neuroendocrinol 11:331–335[CrossRef][Medline]
1-Adrenergic receptor involvement in the LH surge in ovariectomized
estrogen-primed rats. Eur J Pharmacol 81:341–344[CrossRef][Medline]
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A. C. Gore, G. Yeung, J. H. Morrison, and T. Oung Neuroendocrine Aging in the Female Rat: The Changing Relationship of Hypothalamic Gonadotropin-Releasing Hormone Neurons and N-Methyl-D-Aspartate Receptors Endocrinology, December 1, 2000; 141(12): 4757 - 4767. [Abstract] [Full Text] [PDF]
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), c-Fos protein (•), or KA2
receptor subunit and c-Fos (
) or without KA2 receptor subunit and
c-Fos proteins (
) at the peak of the steroid-induced LH surge.
Drawings of coronal sections were taken from five hypothalamic levels
(
200 µm apart) where most GnRH neurons are located: the level of
the vertical limb of the diagonal band of Broca (A), OVLT (B),
anteroventral periventricular nucleus (C), rostral medial preoptic
nucleus (D), and caudal medial preoptic nucleus (E). A representative
animal (CR10) was taken from the group of animals that were killed at
1600 h.
