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Kastor Neurobiology of Aging Laboratories, Fishberg Research Center for Neurobiology, and Henry L. Schwartz Department of Geriatrics and Adult Development, Mount Sinai School of Medicine, New York, New York 10029
Address all correspondence and requests for reprints to: Andrea C. Gore, Ph.D., Neurobiology of Aging Laboratories, Box 1639, Mount Sinai School of Medicine, New York, New York 10029. E-mail: andrea.gore{at}mssm.edu
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Abstract |
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Introduction |
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Alterations in inputs to GnRH neurons from numerous neurotransmitters, growth factors, and steroid hormones probably also play an important role in the regulation of these cells. One neurotransmitter strongly implicated in the regulation of GnRH neurons is glutamate, acting through the N-methyl-D-aspartate (NMDA) receptor (NR) (2, 19, 20, 21) as well as non-NMDA receptors (22, 23) to stimulate GnRH/LH release and biosynthesis. Our laboratory has reported that GnRH neurons of adult female rats express NRs (2), and a similar finding was made by another group (24). Others have found that GnRH neurons express glutamatergic non-NMDA receptors (25). Thus, the effects of glutamate on reproductive function can occur directly on GnRH neurons or be mediated by interneurons.
Age-related alterations in glutamate regulation of neuroendocrine function have recently been reported. NMDA stimulation of GnRH release from hypothalamic explants in vitro and serum LH release in vivo is significantly reduced in aging compared with young female rats (10, 26, 27). It is possible that changes in glutamatergic input to GnRH neurons are a substrate underlying neuroendocrine changes leading to reproductive senescence.
In the present study we performed three experiments to evaluate changes in hypothalamic NRs and their regulation of GnRH neurons during reproductive aging in intact female rats. First, we examined changes in NR subunit mRNAs in POA-AH, the site of GnRH perikarya, and in medial basal hypothalamus (MBH), the site of GnRH axons and neuroterminals, of female rats during reproductive senescence. Second, we tested the ability of NR activation to affect GnRH gene expression during aging. Third, we examined the expression of NRs on GnRH cell bodies of aging rats to determine the anatomical substrate for the regulation of GnRH neurons by NRs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All experiments were conducted in accordance with Guidelines for the Care and Use of Experimental Animals, using protocols approved by the institutional animal care and use committee at Mount Sinai School of Medicine (protocol 96-816NA).
Molecular studies. In Exp I, animals were killed rapidly by decapitation at 1000 or 1500 h (n = 6–8/group). These two time points were chosen based on previous observations of changes in GnRH neurons from 1000–1500 h (3). The rat has a 4- to 5-day estrous cycle comprising proestrus (the day of the preovulatory LH surge, resulting in ovulation), estrus, diestrous day 1, and diestrus day 2. Young animals with regular 4-day cycles and middle-aged rats with regular 4- to 5-day cycles were used on proestrus or diestrus day 1 to select the days of highest and lowest estradiol levels, respectively. It should be noted that in our laboratory, in proestrous rats, the preovulatory LH surge has not yet started at 1500 h in both young and middle-aged rats (3, 18). When rats cease cycling, they enter an acyclic state of persistent estrus or persistent diestrus, and thus acyclic rats were examined on these 2 days. The brain was removed, and the preoptic area-anterior hypothalamus (POA-AH) and medial basal hypothalamus (MBH) were dissected using a stainless steel brain slicer (model RBM-4000, Activational Systems, Warren, MI), snap-frozen on dry ice, and stored at -70 C until use. For these molecular studies, the NR1, NR2a, and NR2b subunit mRNA levels were measured based on previous evidence that these subunits are far more abundant in neuroendocrine regions than the NR2c and NR2d subunits (28), and that NR3 (also called NMDAR-L) subunit expression is undetectable (29) or of low abundance in postnatal brain (30), although this remains to be investigated in hypothalamus.
The POA-AH dissection was made as follows (31, 32). The caudal border was made by a coronal cut just posterior to the entry point of the optic chiasm. The rostral border was exactly 4 mm anterior, made by a coronal cut at the posterior third of the olfactory tubercle. This coronal section (4 mm thick) was laid rostral side up on a chilled glass plate. An isosceles triangle-shaped cut was made with the apex of the triangle just under the midline of the corpus callosum and the two legs of the triangle passing through the anterior commissure. The coronal section from which the MBH dissection was cut had as its rostral border the caudal border of the POA-AH dissection (the point of insertion of the optic nerve into the brain). A second coronal cut was made 3 mm caudal to this cut, at the rostral border of the mammillary bodies. This coronal section (3 mm thick) was laid rostral side up on the glass plate, and another isosceles triangle was cut, with the apex just below the thalamus and the two legs passing through the hypothalamic sulcus at the level of the supraoptic nucleus. Trunk bloods were collected, allowed to clot, centrifuged, and serum stored at -70 C.
In Exp II, to assess effects of N-methyl-D,L-aspartate (NMA) on GnRH gene expression, young and middle-aged rats were implanted with a jugular catheter on diestrus day 1 (cycling rats) or on persistent estrus (acyclic rats) (20). Patency of the catheter was maintained by flushing daily with heparinized saline (20 U/ml). Two days later on proestrus (young and middle-aged cycling rats) or persistent estrus (middle-aged acyclic rats), animals were injected through the catheter with NMA (14 mg/kg in 0.3 ml saline) or vehicle (0.3 ml saline) at 1400 h and killed by decapitation 1 h later at 1500 h. This mode of administration has been used by numerous laboratories (20, 21, 33, 34) and has been reported to cause a rapid activation of GnRH gene expression in young rats (20, 21, 35). Although NMDA is not believed to cross the blood-brain barrier, it is thought to exert its actions directly on hypothalamic regions due to the accessibility of the circumventricular organs to otherwise impermeable substances. A central site of NMDA action is supported by studies demonstrating that NMA does not cause direct release of LH from the anterior pituitary gland (36, 37), and GnRH antagonists block effects of NMA (38, 39). For experiments in the present study, six to eight animals were used per group. The brain was removed, and tissues were dissected as described above.
Microscopic studies. In Exp III, to determine the presence of the NMDA-R1 (NR1) subunit on GnRH neurons, animals at proestrus (young and middle-aged) or persistent estrus (middle-aged) were deeply anesthetized with ketamine (0.35 ml)/xylazine (0.35 ml) and perfused transcardially with 1% paraformaldehyde for 1 min followed by 4% paraformaldehyde for 10 min. Perfusions were performed between 1500–1600 h. The brain was immediately removed and postfixed in 4% paraformaldehyde for 6 h, followed by storage in PBS with 0.5% sodium azide at 4 C. Brains were cut on a Vibratome (Ted Pella, Inc.) at 40 µm and stored in PBS with 0.5% sodium azide at 4 C before processing by immunocytochemistry. Ten animals were used for each group.
Procedures
RNA extraction and ribonuclease (RNase) protection assay.
RNA was extracted from frozen POA-AH dissections as described
previously (20, 40). Cytoplasmic RNA from individual
POA-AH dissections was divided into 5-µg aliquots, each of which was
suspended in 20 µl hybridization solution (0.1 M EDTA, pH
8, and 4 M guanidine thiocyanate; final pH 7.5) for RNase
protection assay. The following DNA clones were used as probes: 1) NR1
mRNA in the cytoplasm was measured using a complementary DNA (cDNA)
clone complementary to 284 bp of the N-terminus, spanning the
BamHI and HindIII restriction sites and subcloned
into a Bluescript KS+ vector (provided by Dr.
Stuart Sealfon, Mount Sinai Medical Center, New York, NY)
(2); 2) NR2a and 3) NR2b mRNAs in the cytoplasm were
measured using cDNA clones complementary to bases 1585–2154 and
1423–1992, respectively, cloned into the SmaI site of the
pBluescript II SK+ vector (provided by Drs.
S. A. Lipton and N. J. Sucher) (29, 41); and 4)
cyclophilin (1B15) was measured using an 111-bp cDNA clone, spanning
from the PstI and XmnI restriction sites and
subcloned in a Bluescript KS+ vector
(31).
Solution hybridization/RNase protection assay was performed as
described previously (20, 40). Briefly, the NR1 and
cyclophilin probes were labeled to low specific activity (
60,000
cpm/ng) and the NR2a and NR2b probes to high specific activity
(1,300,000 cpm/ng) with [
-32P]UTP. The
probes were brought to a final volume of 5 µl and added to samples
and standards (in a volume of 20 µl) for a final volume of 25 µl.
One 5-µg aliquot of each cytoplasmic sample was incubated with the
NR1 and cyclophilin probes in the same tube together with another
transcript, GnRH, that was analyzed in a parallel study
(18). The cyclophilin autoradiograms were presented in
that paper, and no significant differences in cyclophilin mRNA levels
were detectable by reproductive status or age, here and below. Another
5-µg aliquot of each cytoplasmic sample was incubated with the NR2a,
NR2b, and cyclophilin probes in the same tube. For standard curves,
probes were mixed with increasing known amounts of reference RNAs.
Samples and standards were allowed to hybridize for 16–18 h at 30 C;
the remainder of the assay was conducted exactly as described
previously (20, 40). Gels were exposed to x-ray film for
18–48 h to produce an autoradiogram, and to a phosphorimaging screen
(Molecular Dynamics, Inc., Sunnyvale, CA) for 48 h
for quantitation. The amount of radioactivity in each sample was
determined by comparison with the amount of reference RNA calculated by
regression analysis.
Immunocytochemistry. Double label immunofluorescence was performed using the mouse monoclonal antibody to NR1 (54.1; 7.2 mg/ml; 1:750) (42, 43) used in conjunction with the rabbit polyclonal antibody to GnRH (HU60; 1:1000; provided by Dr. Henryk Urbanski) (44). Three tissue sections per animal were chosen at the level of the organum vasculosum of the lamina terminalis (OVLT)/POA, and sections were matched across animals. The procedure was the same as that described previously (45); briefly, tissues were washed three times in PBS for 10 min each time, preincubated in 2% normal goat serum/2% normal horse serum for 1 h, then incubated in primary antibody for 48 h at 4 C. Then, tissues were washed three times for 10 min each time and incubated in secondary antibody [Texas Red-conjugated goat antirabbit IgG (H+L) for HU60, and fluorescein isothiocyanate-conjugated horse antimouse IgG (H+L) for 54.1, both at 1:200] for 1.5 h. Tissues were washed three times for 10 min each time, mounted on gelatin-subbed slides, dried overnight, and coverslipped with Vectashield (Vector Laboratories, Inc., Burlingame, CA). Control sections were incubated in the absence of primary antibody or, in the case of GnRH, were preabsorbed with antigen before immunocytochemistry. In all controls, no specific staining was observed.
Microscopy and analyses. Initially, all sections were examined using a Carl Zeiss Axiophot microscope (New York, NY) at x20 to detect the presence of GnRH neurons, and these cells were mapped for each animal. GnRH neurons were easily detectable and exhibited the classical ovoid or round morphology of GnRH neurons; they were all unipolar or bipolar. Then, each section was examined using a confocal laser scanning microscope (Carl Zeiss LSM 410 inverted confocal microscope) on blind coded immunolabeled sections that were imaged with a Plan-Apochromat x63/1.25 N.A. oil immersion objective at a zoom factor of x3.5 to determine double labeling of NR1 on GnRH neurons. All GnRH neurons were identified, and the percentage of neurons that were double labeled for NR1 was determined in each animal. For four rats (two young, one middle-aged cycling, and one middle-aged acyclic), no GnRH neurons were counted, or there was a technical problem with the immunocytochemistry resulting in no staining, so data from these animals were not included in the analyses. Thus, a total of eight or nine animals per group were used for analyses.
RIA of estradiol. Estradiol levels in serum samples were determined by RIA of duplicate samples using the DSL ultrasensitive estradiol RIA kit (DSL-4800, Diagnostic Systems Laboratories, Inc., Webster, TX) according to the instructions. The assay sensitivity was 5 pg/ml, the interassay variation was 4.6%, and the intraassay variation ranged from 1–3%.
Statistical analyses
For Exp I, differences in NMDA receptor subunit mRNA levels were
analyzed. In all cases, an F test on equality of variances was
performed for the relevant variable. In all cases, this assumption was
met, and therefore, paired t tests were performed among
animals of similar reproductive status but different ages (young and
middle-aged cycling, or middle-aged and old acyclic) to test the
effects of age. Similar analyses were performed on rats of the same age
but different reproductive status (middle-aged cycling and acyclic).
Then, relevant post-hoc comparisons were made using
Fisher’s protected least significant difference (PLSD) analysis.
Because no significant effect of time of day was found for any of the
parameters examined, data from the 1000 and 1500 h points were
combined for all analyses. For Exp II, effects of NMA or vehicle on
GnRH gene expression were assessed by two-way ANOVA (variables: age and
reproductive status), followed by Fisher’s PLSD analysis. In Exp III,
differences in labeling of GnRH neurons with the NR1 were tested by
ANOVA as in Exp II. As in Exp I, post-hoc comparisons were
performed using Fisher’s PLSD analysis. In all cases, significance was
set at P < 0.05. Because of the large number of
statistical comparisons made, in Results we have focused on
significant changes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The
only significant difference in NR1 mRNA levels in POA-AH was observed
in acyclic persistent estrous rats, in which NR1 mRNA levels were
significantly higher in old rats than in their middle-aged counterparts
(P < 0.05; Fig. 2).
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. Overall, NR1 mRNA levels
increased with age in the MBH (P < 0.0001). NR1 mRNA
levels were higher in middle-aged diestrus day 1 than in young
diestrous day 1 rats (P < 0.05; Fig. 2
). Levels of NR1
mRNA were maintained at this level in acyclic middle-aged and old rats,
with no differences between these groups. Additionally, a significant
effect of reproductive status was found in middle-aged rats
(P < 0.05), with higher NR1 mRNA in middle-aged
persistent estrous and persistent diestrous compared with proestrous
rats (P < 0.05; Fig. 2).
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.
NR2a mRNA levels decreased during aging. In particular, this decrease
occurred on the basis of reproductive status, with acyclic rats having
significantly lower NR2a mRNA levels than cycling rats
(P < 0.005; Fig. 5). In
acyclic rats, on persistent diestrus, old rats had higher NR2a mRNA
levels than middle-aged rats (P < 0.05; Fig. 5).
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. In contrast to the
POA-AH, NR2a mRNA levels in the MBH increased during aging (Fig. 5). In
cycling rats, NR2a mRNA levels were significantly higher in middle-aged
diestrous day 1 rats than in middle-aged proestrous or young rats of
either cycling status (P < 0.05; Fig. 5). The higher
levels in middle-aged diestrous day 1 rats were maintained in acyclic
rats at middle-aged and old ages (Fig. 5).
Changes in NR2b subunit mRNA with age and reproductive status
POA-AH. A representative autoradiogram showing NR2b mRNA in
representative POA-AH dissections of rats is shown in Fig. 4. NR2b mRNA
levels in POA-AH differed on the basis of reproductive status, with
acyclic rats having lower levels than cycling rats (Fig. 6). Although NR2b mRNA levels were
significantly lower in middle-aged acyclic than in cycling rats
(P < 0.005; Fig. 6), levels increased slightly in old
persistent diestrous rats, which had higher levels than the
corresponding middle-aged group (P < 0.05; Fig. 6).
Thus, both NR2a and NR2b mRNA levels in the POA-AH, the site of GnRH
perikarya, decreased on the basis of age and, more particularly, on the
basis of reproductive status.
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. NR2b mRNA
levels in the MBH increased with aging (P < 0.05; Fig. 6). A significant effect of reproductive status was found
(P < 0.005), with middle-aged proestrous rats having
significantly lower NR2b mRNA levels than middle-aged persistent
estrous or persistent diestrous rats (P < 0.005; Fig. 6). Levels of NR2b mRNA in middle-aged acyclic rats were maintained in
the old group. Thus, both NR2a and NR2b mRNA levels in the MBH, the
site of GnRH axons and neuroterminals, increased during reproductive
aging, with differences on the basis of both age and reproductive
status.
Exp II: effects of NMA on GnRH mRNA levels in aging rats
The results of Exp I indicate that NR mRNA levels in the POA-AH
differ primarily on the basis of reproductive status, as opposed to
absolute chronological age. Therefore, we investigated the effects of
NR activation on GnRH mRNA levels in young proestrous, middle-aged
proestrous, and middle-aged persistent estrous female rats. It has been
reported that injection of NMA into young rats causes a rapid increase
in LH release, which is strongly reduced or abolished in middle-aged
and old rats (26, 27). In the present study, NMA was
administered, and animals were killed 1 h later. The effects of
NMA on GnRH mRNA levels were determined by RNase protection assay. GnRH
mRNA levels were significantly altered 1 h after NMA treatment
(P < 0.0001). GnRH mRNA levels in young proestrous
rats were significantly stimulated 1 h after NMA treatment
(P < 0.005; Fig. 7). In
contrast, in middle-aged proestrous and persistent estrous rats, NMA
caused a significant decrease in GnRH mRNA levels (P <
0.005; Fig. 7). Thus, there is an age-dependent switch in the direction
of the response to NR activation, from stimulatory to inhibitory,
between the young and middle-aged groups.
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800–2000 GnRH neurons
are estimated to exist in the rodent brain) (46, 49), a
relatively small number of GnRH-positive perikarya were identified in
the present study. However, this number provided sufficient statistical
power to perform statistical analyses. A photomicrograph of GnRH cell
bodies from young and middle-aged rats, double labeled for NR1, is
shown in Fig. 8. The quantitation of
expression of NR1 in GnRH neurons is presented in Table 1. A trend toward a decrease in the
number of GnRH neurons was seen between young and middle-aged rats.
However, this was not significant (P = 0.11) due to
considerable variability in the number of GnRH neurons detected between
animals; nevertheless, this finding is consistent with other reports
demonstrating no differences or small decreases in the numbers of GnRH
neurons with aging (50, 51). The percentage of GnRH somata
expressing NR1 was found to differ among the three groups examined
(P < 0.05), with significantly lower double
labeling in middle-aged proestrous than in middle-aged persistent
estrous rats (P < 0.05). A trend for a lower
percentage of double labeling in middle-aged proestrous compared with
young proestrous rats was also seen, although this did not reach
significance (P = 0.12), again probably due to the high
variability between animals within a group.
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As expected, estradiol levels in cycling rats were higher on proestrus
than on diestrous day 1 (P < 0.05). Levels of
estradiol in acyclic rats were similar to those in proestrous rats. The
high estradiol levels in the middle-aged and old acyclic groups are
similar to those reported by other laboratories (52, 53).
The probable explanation for the maintenance of these high levels of
estradiol in aged rats is that this species, unlike humans, does not
undergo follicular atresia at the ages examined in the present
study.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Exp I, we measured NR subunit mRNAs in the POA-AH, the site of GnRH somata. The NR1 subunit is obligatory for a functional NR (41). NR1 mRNA levels varied only in the old persistent estrous rats. Together with our previous finding that NR1 mRNA levels in the POA-AH do not change much after day 10 postnatally (2, 45), this suggests that mRNA levels of this subunit are not the limiting factor for age-related changes in the NR pentamer. However, our data are in contrast to those of another group who reported a decrease in NR1 mRNA in POA of middle-aged compared with young female rats as determined by RT-PCR (10). Differences between our studies are probably due to the use of different techniques, probes, or dissection.
We also measured NR2a and NR2b mRNAs in the POA-AH. These subunits are abundant in hypothalamus, whereas NR2c and NR2d are sparse in these regions (28). As the NR is heteromeric, comprising the NR1 subunit together with at least one member of the NR2 family (41), it is likely that the most abundant heteromer formed in the POA-AH is the NR1 subunit together with the NR2a and/or NR2b subunit. It has been reported that approximately 17% of GnRH neurons express NR2a (28), suggesting that this may be the major subunit combining to form pentamers with the NR1 subunit in POA-AH.
We found that NR2a and NR2b mRNA levels in the POA-AH decreased during reproductive senescence, particularly on the basis of reproductive status, independent of chronological age. We do not know the causality of these phenomena, i.e. whether the decrease in NR2a and NR2b mRNA is a consequence of neuroendocrine senescence, or if changes in NR subunits precipitate or play a role in the transition to acyclicity. Furthermore, the specific cells on which the NRs are located cannot be determined by this method, although this issue was addressed by Exp III. Nevertheless, changes in NR2a and NR2b mRNA levels could have physiological consequences if reflected by similar changes in protein levels. Thus, the decrease in NR2a and NR2b levels in acyclic animals would cause the ratio of NR1 to NR2a and NR2b to be altered such that the relative proportion of NR1 would be higher. This potential change in receptor stoichiometry could have physiological consequences for glutamate binding and signal transduction. The NR1 subunit is obligatory for the function of a NMDA receptor, probably ligand binding, whereas the NR2 subunits play a role in ion gating properties and removal of the Mg2+ block (41). Thus, the binding properties of the NR or the ability of the NR to transduce a signal may change with reproductive aging. An example of the effect of changing receptor stoichiometry is demonstrated by a recent report from one laboratory that overexpressed the NR2b receptor subunit mRNA in mouse forebrain. This profoundly affected the electrophysiological properties of hippocampal neurons and NR-mediated functions involved in learning and memory (54).
NR subunit gene expression was also measured in the MBH, the site of GnRH axons and neuroterminals. All three NR subunit mRNAs increased with chronological age and on the basis of reproductive status, with higher levels in acyclic rats. This result differs from that of another laboratory (10), which found that NR1 mRNA levels, measured by RT-PCR, decreased in the arcuate nucleus/median eminence of middle-aged vs. young proestrous rats. Again, differences may be attributable to technical issues. Our results in the MBH, as in the POA-AH, are consistent with the likelihood of altered stoichiometries of NRs and correspondingly differing functional properties during reproductive aging. Unlike the POA-AH, the direction of this age-related change in the MBH is an increase, and with respect to stoichiometry, it is likely that the ratio of the NR1 to the NR2a and NR2b subunits decreases. However, the functional consequences in such a shift in stoichiometry are currently unknown. Nevertheless, it is puzzling why NR2a and NR2b mRNA levels decrease in the POA-AH and increase in the MBH with reproductive acyclicity. It might be predicted that the regulation of GnRH neurons by glutamate in the POA-AH is primarily on biosynthesis of the decapeptide in the cell body, whereas effects of glutamate at the MBH would be on GnRH neuroterminals and GnRH release. Again, however, it is unknown how these changes in NR subunit gene expression directly relate to GnRH cells per se, as many neuroterminals other than GnRH are present in the MBH and median eminence, some of which are NMDA responsive and some of which may release neurotransmitters in response to NMDA receptor activation that also affect GnRH release.
The results demonstrating age-related changes in hypothalamic NRs are
quite relevant to other hypothalamic neuronal populations. NRs regulate
hypothalamic factors such as CRH (55), MSH
(56), and somatostatin (57). NMDA affects
magnocellular neurons of the supraoptic and paraventricular nuclei
(58, 59); it affects circadian activity in the
suprachiasmatic nucleus (60), and hypothalamic NRs
interact with neurotransmitters such as serotonin (61) and
-aminobutyric acid (62). The changes in
hypothalamic NRs observed in the present study would be expected to
impact upon these systems as well as GnRH neurons. Moreover, a strong
relationship between NMDA receptors and estrogen has been reported for
GnRH as well as other neurons. Most relevant to the present study is
the enhanced ability of NMDA to stimulate GnRH in the presence of
estrogen (19) and the expression of estrogen receptors in
glutamate neurons in the monkey (63). Although the rats in
the present study continue to have elevated estradiol levels during
aging, it is possible that the exposure of the brain to cyclic as
opposed to continuous levels of estradiol could alter the functional
properties of NRs.
The latter observations are also relevant to nonhypothalamic regions. Dendritic spine density in the CA1 region of hippocampus is affected by estradiol, and this is a NR-dependent phenomenon (64). Thus, changes in NR mRNA levels and alterations in the stoichiometry of the NR could be predicted to occur in the hippocampus and other regions, particularly because changes in learning, memory, cognitive function, and long-term potentiation (LTP) involve NRs (65), and become compromised during aging (66). Our laboratory has conducted a parallel set of studies on measurements of NR mRNAs in the hippocampus of intact rats during aging (67). The most intriguing finding was that alterations in NR mRNAs in hippocampus occurred during the transition from middle-aged to aged, the exact period associated with a significant decline in cognitive function (66). In contrast, the alterations in hypothalamic NRs are primarily associated with the transition to reproductive senescence during the middle-aged period. Thus, alterations in NR mRNAs in hippocampus and hypothalamus occur at different life transitions and appear to be associated with functional end points such as cognitive or reproductive decline, respectively.
In Exp II we examined the regulation of GnRH neurons by NR activation, by administering NMA to young proestrous, middle-aged proestrous, and middle-aged persistent estrous rats and measuring its effect on GnRH mRNA levels. It has previously been demonstrated that GnRH mRNA levels in young rats are significantly elevated by 15 min after NMA treatment (20, 21). The present results demonstrate an age-related switch in the direction of the effects of NMA on GnRH gene expression; although NMA stimulated GnRH mRNA levels in young proestrous rats, it depressed GnRH mRNA levels in middle-aged rats regardless of reproductive status. These data are consistent with reports of age-related decreases in the ability of NMDA to stimulate GnRH neurons. NMDA-induced GnRH release in vitro was reduced in middle-aged or old vs. young rats (10, 26, 27). Additionally, intrahypothalamic administration of NMDA caused an increase in serum LH in young, but not aged, ovariectomized rats (26). Other laboratories reported decreases in NMDA receptor binding in rat hypothalamus during aging (68) and decreased levels of hypothalamic glutamate (27). These studies suggest that the ability of NMDA to activate GnRH neurons becomes compromised during aging.
Regarding the localization of NRs on GnRH neurons, we had previously reported that approximately 20% of GnRH neurons express the NR1 subunit in young, cycling female rats (2). The results of Exp III are comparable, with approximately 30% of GnRH neurons expressing the NR1 subunit in young proestrous rats. A similar finding was recently reported by another group (24), although others have reported smaller percentages or no GnRH neurons colocalizing with NR1 (28, 69), possibly due to technical differences.
Two cellular changes were observed during reproductive aging in the present study. First, the number of detectable GnRH neurons tended to decrease from young to middle-aged rats. Other laboratories have reported that the total number of GnRH remains the same (4, 70, 71) or decreases with aging (50, 51), and the results of our study are consistent with these latter findings. Moreover, our results suggest the possibility that age-related decreases in the number of GnRH neurons may be reproductive status independent. Second, when we calculated the percentage of GnRH neurons expressing the NR1 subunit, we made the unexpected finding of similar percentages in young proestrous and middle-aged persistent estrous rats (levels are somewhat higher in the latter group), but lower percentages in middle-aged proestrous rats. Qualitative microscopic observations indicate that the overall amount of NR1 immunoreactivity did not appear to differ among the three groups, consistent with the mRNA data of Exp I demonstrating no differences in NR1 mRNA levels among these three groups. Thus, alterations in numbers of NR1-expressing cells between middle-aged proestrous and persistent estrous rats appear to be specific to the GnRH-positive population. It is also possible that the overall number of GnRH neurons does not change, but that the ability to detect these neurons by immunocytochemistry is altered during reproductive aging; however, that cannot be determined in the present study.
The meaning of the high levels of double labeling in the middle-aged persistent estrous rats is unknown. It is possible that it is a compensatory mechanism by which an animal responds to lack of fluctuating estrogen levels across the estrous cycle, although further studies would be required to demonstrate this phenomenon. The present result is particularly puzzling in light of Exp II, which demonstrates that treatment with NMA stimulates GnRH gene expression in young and inhibits it in middle-aged rats regardless of reproductive status. Therefore, the anatomical colocalization of the NR1 subunit on GnRH neurons does not correlate with this functional response. It is possible that the inhibitory response to NMA in middle-aged rats may be due to different mechanisms in cycling and acyclic animals. In middle-aged proestrous rats, it may be related to the change in double labeling of the NR1 subunit on GnRH neurons, and in middle-aged persistent rats it may be due to the changes in NR2a and NR2b subunits. Additionally, apparent differences between groups may be attributable to differences in translation of the NR subunits to protein; thus, although NR2a and 2b mRNA levels may decrease in middle-aged persistent estrous rats, this may not be reflected by parallel changes in protein levels. Alternatively, the changes in NR2a and 2b mRNA levels measured in Exp I are throughout the POA-AH and may not be specific to GnRH neurons. These issues could be addressed by immunocytochemical analyses of expression of the NR2a and NR2b subunits on GnRH neurons, experiments which are currently underway.
In summary, the present study demonstrates molecular, physiological, and cellular changes in hypothalamic NRs and their interactions with GnRH neurons, which may play a role in the transition to acyclicity in aging female rats. Many of these changes appear to occur on the basis of reproductive cyclicity, not absolute chronological age, and demonstrate the need to consider the function of NRs on this basis. Future studies will attempt to determine whether such changes in NRs are causal for reproductive senescence or whether they are a consequence of other neurobiological changes that occur during aging. The role of estrogen in this phenomenon will also be elucidated.
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Acknowledgments |
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Footnotes |
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Received April 12, 2000.
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References |
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-MSH from the rat hypothalamus through release of nitric
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  G. S. Neal-Perry, G. D. Zeevalk, J. Shu, and A. M. Etgen Restoration of the Luteinizing Hormone Surge in Middle-Aged Female Rats by Altering the Balance of GABA and Glutamate Transmission in the Medial Preoptic Area Biol Reprod, November 1, 2008; 79(5): 878 - 888. [Abstract] [Full Text] [PDF]
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 Q.-G. Zhang, R. Wang, M. Khan, V. Mahesh, and D. W. Brann Role of Dickkopf-1, an Antagonist of the Wnt/{beta}-Catenin Signaling Pathway, in Estrogen-Induced Neuroprotection and Attenuation of Tau Phosphorylation J. Neurosci., August 20, 2008; 28(34): 8430 - 8441. [Abstract] [Full Text] [PDF]
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 W. Yin, J. M. Mendenhall, S. B. Bratton, T. Oung, W. G. M. Janssen, J. H. Morrison, and A. C. Gore Novel Localization of NMDA Receptors Within Neuroendocrine Gonadotropin-Releasing Hormone Terminals Experimental Biology and Medicine, May 1, 2007; 232(5): 662 - 673. [Abstract] [Full Text] [PDF]
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 B. M. Nathan, C. A. Hodges, P. J. Supelak, L. C. Burrage, J. H. Nadeau, and M. R. Palmert A Quantitative Trait Locus on Chromosome 6 Regulates the Onset of Puberty in Mice Endocrinology, November 1, 2006; 147(11): 5132 - 5138. [Abstract] [Full Text] [PDF]
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J. H. Morrison, R. D. Brinton, P. J. Schmidt, and A. C. Gore Estrogen, Menopause, and the Aging Brain: How Basic Neuroscience Can Inform Hormone Therapy in Women J. Neurosci., October 11, 2006; 26(41): 10332 - 10348. [Full Text] [PDF]
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M. El-Etr, Y. Akwa, E.-E. Baulieu, and M. Schumacher The Neuroactive Steroid Pregnenolone Sulfate Stimulates the Release of Gonadotropin-Releasing Hormone from GT1-7 Hypothalamic Neurons, through N-Methyl-D-Aspartate Receptors Endocrinology, June 1, 2006; 147(6): 2737 - 2743. [Abstract] [Full Text] [PDF]
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 W. Yin and A. C Gore Neuroendocrine control of reproductive aging: roles of GnRH neurons. Reproduction, March 1, 2006; 131(3): 403 - 414. [Abstract] [Full Text] [PDF]
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G. S. Neal-Perry, G. D. Zeevalk, N. F. Santoro, and A. M. Etgen Attenuation of Preoptic Area Glutamate Release Correlates with Reduced Luteinizing Hormone Secretion in Middle-Aged Female Rats Endocrinology, October 1, 2005; 146(10): 4331 - 4339. [Abstract] [Full Text] [PDF]
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 R. G. Smith, L. Betancourt, and Y. Sun Molecular Endocrinology and Physiology of the Aging Central Nervous System Endocr. Rev., April 1, 2005; 26(2): 203 - 250. [Abstract] [Full Text] [PDF]
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T. R. Chakraborty and A. C. Gore Aging-Related Changes in Ovarian Hormones, Their Receptors, and Neuroendocrine Function Experimental Biology and Medicine, November 1, 2004; 229(10): 977 - 987. [Abstract] [Full Text] [PDF]
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A. C. Gore, B. M. Windsor-Engnell, and E. Terasawa Menopausal Increases in Pulsatile Gonadotropin-Releasing Hormone Release in a Nonhuman Primate (Macaca mulatta) Endocrinology, October 1, 2004; 145(10): 4653 - 4659. [Abstract] [Full Text] [PDF]
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 A. Liu, Z. Zhuang, P. W. Hoffman, and G. Bai Functional Analysis of the Rat N-Methyl-D-aspartate Receptor 2A Promoter: MULTIPLE TRANSCRIPTION START POINTS, POSITIVE REGULATION BY Sp FACTORS, AND TRANSLATIONAL REGULATION J. Biol. Chem., July 11, 2003; 278(29): 26423 - 26434. [Abstract] [Full Text] [PDF]
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T. R. Chakraborty, L. Ng, and A. C. Gore Colocalization and Hormone Regulation of Estrogen Receptor {alpha} and N-Methyl-D-Aspartate Receptor in the Hypothalamus of Female Rats Endocrinology, January 1, 2003; 144(1): 299 - 305. [Abstract] [Full Text] [PDF]
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E. N. Ottem, J. G. Godwin, and S. L. Petersen Glutamatergic Signaling through the N-Methyl-D-Aspartate Receptor Directly Activates Medial Subpopulations of Luteinizing Hormone-Releasing Hormone (LHRH) Neurons, But Does Not Appear to Mediate the Effects of Estradiol on LHRH Gene Expression Endocrinology, December 1, 2002; 143(12): 4837 - 4845. [Abstract] [Full Text] [PDF]
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B. H. Miller and A. C. Gore N-Methyl-D-Aspartate Receptor Subunit Expression in GnRH Neurons Changes during Reproductive Senescence in the Female Rat Endocrinology, September 1, 2002; 143(9): 3568 - 3574. [Abstract] [Full Text] [PDF]
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J. Hou, B. Li, Z. Yang, N. Fager, and M. Y. J. Ma Functional Integrity of ErbB-4/-2 Tyrosine Kinase Receptor Complex in the Hypothalamus Is Required for Maintaining Normal Reproduction in Young Adult Female Rats Endocrinology, May 1, 2002; 143(5): 1901 - 1912. [Abstract] [Full Text] [PDF]
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J. Hou, B. Li, Z. Yang, N. Fager, and M. Y. J. Ma Altered Gene Activity of Epidermal Growth Factor Receptor (ErbB-1) in the Hypothalamus of Aging Female Rat Is Linked to Abnormal Estrous Cycles Endocrinology, February 1, 2002; 143(2): 577 - 586. [Abstract] [Full Text] [PDF]
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 P. M. Wise, M. J. Smith, D. B. Dubal, M. E. Wilson, S. W. Rau, A. B. Cashion, M. Bottner, and K. L. Rosewell Neuroendocrine Modulation and Repercussions of Female Reproductive Aging Recent Prog. Horm. Res., January 1, 2002; 57(1): 235 - 256. [Abstract] [Full Text] [PDF]
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