Attenuation of Preoptic Area Glutamate Release Correlates with Reduced Luteinizing Hormone Secretion in Middle-Aged Female Rats
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《内分泌学杂志》
Department of Obstetrics and Gynecology, Division of Reproductive Medicine and Infertility (G.S.N.-P., N.F.S.) and Department of Neuroscience (A.M.E.), Albert Einstein College of Medicine, Bronx, New York 10461
Levine Neuroscience Laboratory, Department of Neurology (G.D.Z.), University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medial School, Piscataway, New Jersey 08854
Abstract
Glutamate (Glu) and its receptors are involved in the maturation and maintenance of the neural mechanisms governing the preovulatory LH surge of young, reproductive-aged rodents and nonhuman primates. Little is known about the role of Glu in the delayed onset and reduced peak amplitude of the LH surge that characterizes female rodents during early reproductive senescence. The present study tested the hypothesis that the delayed and attenuated LH surge observed in middle-aged female rats is associated with altered hypothalamic Glu release. We used intracerebral microdialysis in young (3–4 months) and middle-aged (9–11 months) female rats to monitor changes in medial preoptic area Glu release and jugular vein catheters to monitor changes in serum LH levels. All animals were ovariectomized and injected with estradiol and progesterone in doses sufficient to produce a robust LH surge in most (70%) young rats. In both young and middle-aged females that surged, extracellular Glu levels were higher than in those that did not surge. Among animals that surged, the onset of the LH surge was significantly delayed, and the amplitude of the surge was significantly reduced in middle-aged compared with young rats. Middle-aged females also had significantly reduced extracellular Glu levels throughout the day of the LH surge when compared with young females. These data strongly suggest that age-related hypothalamic dysfunction contributes to reproductive aging independent of gonadal failure. We propose that reduced medial preoptic area Glu transmission contributes to reproductive aging by attenuating excitatory input to GnRH neurons.
Introduction
REPRODUCTIVE AGING IN female nonhuman primates and rodents is characterized by alterations in the patterns of LH and follicle-stimulating hormone release (1, 2, 3, 4, 5, 6, 7), especially in the periovulatory period. It has been postulated that the age-related decline in functional ovarian reserve (3, 4, 7, 8, 9) and coincident hypoestrogenic and hypoprogestinemic hormonal milieu trigger secondary changes in gonadotropin release patterns. Together, these events were believed to initiate the transition into reproductive quiescence (3, 5, 9, 10). Although it is clear that ovarian failure ultimately defines human reproductive quiescence (11), a growing number of studies implicate a significant neuroendocrine component to reproductive aging in primate and nonprimate models (4, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21).
In rodents, a delayed onset and attenuated amplitude of the preovulatory LH surge heralds the onset of reproductive senescence (18, 21). This change in LH release begins to occur when rodents are 8–12 months old, an age equivalent to human middle age (21, 22). Changes in rodent gonadotropin release patterns precede other indicators of reproductive senescence, such as irregular estrous cyclicity. These age-related changes in gonadotropin release do not correlate with reduced basal GnRH peptide release (20, 23, 24), reduced capacity for activated GnRH neurons to release GnRH (23, 25), or reduced GnRH neuronal density (26, 27). Moreover, alterations in LH release from middle-aged cycling rats do not result from abnormal pituitary responsiveness to estrogen positive feedback (28, 29) or GnRH peptide (22, 23, 28, 29). Age-related changes in gonadotropin release have, however, been associated with a 50% reduction in GnRH neuronal activation when c-Fos was used as a marker for cellular activation (30) and with reduced mediobasal hypothalamic GnRH release on the day of proestrus (31). Similarly, altered circadian rhythms of norepinephrine (NE) turnover (32) and release (15) and attenuated vasoactive intestinal peptide mRNA (33) levels have been reported in specific hypothalamic nuclei of middle-aged rats.
Maturation of the GnRH neuronal network during the pubertal transition and hypothalamic control of estrous cyclicity are thought to involve changes in the balance between excitatory input from glutamatergic and inhibitory input from GABAergic neuronal processes (34, 35, 36, 37, 38, 39, 40, 41). In mature, steroid-primed female rats, increases in hypothalamic glutamate (Glu) release (42) and Glu receptor activation stimulate GnRH neurons (43) and induce GnRH peptide synthesis and release (44, 45) and, consequently, pituitary LH synthesis and release. The preovulatory LH surge is preceded by an increase in extracellular Glu levels (42, 44, 46, 47, 48) in the hypothalamic medial preoptic area. The Glu receptor agonist, N-methyl-D-aspartic acid (NMDA), stimulates GnRH peptide synthesis and secretion and advances the estradiol (E2) and progesterone (P)-induced LH surge (47, 48). Likewise, the NMDA receptor antagonist, MK801, blocks the E2- and P-induced LH surge (47, 48, 49). Although increases in hypothalamic Glu levels and Glu receptor activation are implicated in the regulation of GnRH neuronal activation and GnRH synthesis and release in young rats, the status of hypothalamic Glu neurotransmission and its possible role in the initiation and maintenance of reproductive senescence has received little attention (50, 51, 52, 53). We therefore tested the hypothesis that age-related changes in hypothalamic Glu release contribute to age-related alterations in LH release. We used intravenous jugular vein catheters for serum collection and intracerebral microdialysis in the hypothalamic medial preoptic area of young and middle-aged rats that were ovariectomized and primed with E2 plus P to study the magnitude of extracellular Glu levels attained on the day of the LH surge and the temporal relationships among the attainment of maximal medial preoptic area Glu levels and the onset and amplitude of the LH surge.
Materials and Methods
Animals
Young (3–4 months) and middle-aged (9–11 months) female Sprague Dawley rats were purchased from Taconic Farms (Germantown, NY). All rats had access to food and water ad libitum and were housed individually and maintained on a 14-h light, 10-h dark cycle with lights off at 2000 h. Only those rats that exhibited at least three normal 4-d estrous cycles, as determined by daily vaginal smears, were used for microdialysis and LH release studies.
Stereotaxic surgery and jugular vein catheterization
Young (n = 14) and middle-aged (n = 16) rats that exhibited normal estrous cycles were anesthetized with im ketamine (80 mg/kg) and xylazine (4 mg/kg) for ovariohysterectomy (54) and intracerebral microdialysis guide cannula placement. Anesthetized rats were placed in a Kopf stereotaxic apparatus and secured with ear bars and a nose piece set at +5 mm. Using Bregma as a landmark and stereotaxic coordinates provided in the atlas of Pellegrino et al. (55) (dorsal/ventral, –8.5; anterior/posterior, +2.0; and medial/lateral, ±0.6), a unilateral 23-gauge guide cannula was implanted in the hypothalamic medial preoptic area. Guide cannulae and concentric dialysis probes (2-mm dialysis surface with 340-μm outer diameter) were purchased from Bioanalytical Systems, Inc. (West Lafayette, IN). All rats were allowed to recover for 7 d. On the 8th postoperative day, rats with intracerebral microdialysis guide cannulae were lightly anesthetized with ketamine/xylazine and an internal jugular vein catheter placed in the right atrium for serial blood sampling (56). Catheters were kept patent with daily infusion of 1 ml heparinized saline (50 IU). All studies were carried out according to protocols approved by the Institutional Animal Care and Use Committee of Albert Einstein College of Medicine and according to National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Gonadal steroid priming
To ensure that young and middle-aged females were exposed to equivalent hormone levels, gonadal steroid priming for the induction of the LH surge was achieved with previously described methods (57). At 0900 h on the day of catheterization, rats were injected sc with 2 μg E2 benzoate (in 0.1 ml peanut oil); a second injection was given 24 h later. To augment the LH surge, a single sc injection of 500 μg P was given at 0900 h, 48 h after the first E2 injection (58). This hormone treatment produced LH surges in eight of 11 young and eight of 12 middle-aged rats.
Microdialysis sampling and serum collection
To avoid acute injury-induced fluctuations in neurotransmitter release at the time of intracerebral microdialysis probe placement (39), probes were lowered at least 12 h before the start of each experiment. Microdialysis sample collection was initiated 1 h before the P injection. The probes were perfused at a rate of 1.25 μl/min with artificial cerebral spinal fluid [124 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 1.8 mM CaCl2, 26 mM NaCO3, and 7.2 mM dextrose (pH 7.4)]. Microdialysis samples were collected from anesthetized, freely moving rats for a total of 13 h at 30-min intervals. All microdialysis samples were acidified to 0.1 N perchloric acid, flash frozen, and stored at –70 C until analysis with HPLC. Blood sampling was initiated at the time of the P injection and continued every 1–2 h for a total of 12 h. Approximately 200 μl blood was collected into Eppendorf tubes containing 100 μl ice-cold heparinized saline (10 IU), refrigerated overnight, and centrifuged at 1000 x g for 10 min. Serum was removed with a glass pipette and stored at –70 C until assayed for LH. At the end of each experiment, animals were killed with halothane, decapitated, and the brain rapidly dissected and frozen in 2-methylbutane. Frozen brains were stored at –70 C until cryostat sectioning for histological assessment of probe placement.
LH assay
Serum LH levels were determined in duplicate with a rat double antibody LH enzyme immunoassay (Biotrak System, Amersham Bioscience Corp., Piscataway, NJ). The lower limit of the assay was 0.1 ng/ml, and the intra- and interassay coefficients of variation were 8.4 and 11.5%, respectively.
E2 and P assays
Sera collected 2 h after the P injection were used to determine serum P and E2 levels by fluoroimmunoassay using the DELFIA (PerkinElmer Life Sciences, Turku, Finland) E2 and P assays. All sample measurements were performed in duplicate, and the mean value was reported. The lower limit for E2 detection was 13.6 pg/ml, and the intra- and interassay coefficients of variance were 6.1 and 5.8%, respectively. The lower limit for P detection was 0.25 ng/ml, and the intra- and interassay coefficients of variance were 4.9 and 7.0%, respectively.
HPLC analysis of Glu
Amino acid content in medial preoptic area microdialysis samples was quantified by methods previously described (59, 60). Briefly, microdialysis samples were adjusted to pH 5 with 2.5 M potassium carbonate. After o-phthalaldehyde derivatization, amino acids were separated by gradient elution and detected with a Beckman 175 fluorometer. Derivatized samples were applied to a C-18 column (0.4- x 2.5-cm reverse phase, Beckman 5-μm Ultrasphere) at a flow rate of 0.75 ml/min. A programed gradient elution was created with dual HPLC pumps and two solvent mixtures [solvent A, 0.1 M sodium acetate (pH 5.9) in 10% methanol; solvent B, 80% methanol]. Amino acid identification and quantification were achieved by comparing peak retention times and heights of known standards (Sigma, St. Louis, MO) to unknown samples. Amino acid content is reported as picomoles per microliter. The lower limit for detection of Glu is 0.05 pmol/μl.
Histological verification of probe placements
Frozen brains were sectioned in the transverse plane with a cryostat. Every third 40-μm section was saved throughout the extent of the dialysis probe track, stained with thionin, and inspected for confirmation of intracerebral microdialysis probe placement in the medial preoptic area. Only those rats in which probe placement was confirmed were included in the data analysis. Three rats from each age group were discarded due to inaccurate probe placement.
LH surge analysis
A LH surge was defined as an increase in serum LH levels equal to or greater than 1.5 times baseline levels for a minimum of two samples. Baseline LH levels were defined as the LH value observed at the time of the P injection. The LH surge onset was considered delayed if it occurred at a time greater than 2 SD values from the mean onset time of young rats included in our studies. Because our objective was to determine whether the delayed onset and reduced peak amplitude of the LH surge in middle-aged rats was associated with age-related differences in medial preoptic area Glu levels, certain analyses only included data from middle-aged rats that exhibited a delayed and attenuated LH surge and young rats with a normal LH surge (defined as a LH surge with an onset and peak amplitude that falls within 2 SD values of mean values for all young rats with surges). Only one of nine middle-aged rats failed to meet our criterion for a delayed onset of the LH surge and was therefore excluded from data analysis. Only one of eight young rats failed to mount a LH surge within 2 SD values of mean values for all young rats with surges. This rat exhibited a LH surge with a delayed onset and reduced amplitude and was therefore excluded from our data analysis.
Statistical analysis
The integrated area under the curve (AUC) for total medial preoptic area Glu levels and total serum LH release was calculated using Pharm/PCS version 4.2 (61). All data are expressed as mean ± SE. Two-way ANOVA was used to evaluate age-related differences between the AUC of serum LH and medial preoptic area Glu levels in rats with and without a LH surge. Differences in mean peak LH release, pre-LH surge Glu AUC, Ser AUC, mean peak Glu release, and the time of the onset of the LH surge relative to the time of P injection were analyzed with Student’s t tests. Two-way ANOVA was used to compare differences in body weight, plasma E2, P, and baseline LH levels in young and middle-aged rats with and without LH surges. P 0.05 was accepted as statistically significant. A Fisher exact test was used to evaluate age-related differences in the time of peak Glu levels relative to the onset of the LH surge.
Results
Middle-aged rats exhibit a delayed onset and attenuated peak amplitude of the LH surge
Baseline LH values at the time of P injection are equivalent in young and middle-aged rats (Table 1). Two hours after the P injection, gonadal steroid levels were also equivalent in young and middle-aged rats despite their differences in body weight (Table 1). Similar percentages of young (72%) and middle-aged (69%) rats with verified probe placements mounted a LH surge with our E2 and P protocol. Most middle-aged rats (eight of nine; 89%) exhibited a delayed onset and attenuated peak amplitude of the LH surge. Most young rats (seven of eight; 88%) mounted a robust LH surge that begins between 4–5 h after P injection. When compared with young rats, middle-aged rats release half as much LH (P < 0.05) on the day of the steroid-induced LH surge (Table 2 and Fig. 1). Similarly, the mean peak amplitude of LH release in individual middle-aged rats is reduced by approximately 60% (P < 0.01) compared with young rats (Table 3 and Fig. 2). When a LH surge failed to occur, serum LH levels in middle-aged and young rats were equivalent (Table 2). When compared with young rats exhibiting a LH surge, the onset of the LH surge in middle-aged rats was delayed by approximately 3 h (P < 0.01) (Table 3 and Fig. 2). These results are in agreement with others (31, 62).
The LH surge is associated with higher extracellular levels of medial preoptic area Glu
Young and middle-aged rats that have a LH surge have 2-fold higher levels of extracellular Glu in the medial preoptic area than animals of the same age that fail to surge (Figs. 3 and 4). Thus, the LH surge in both young and middle-aged rats is associated with higher levels of extracellular Glu in the medial preoptic area (P < 0.05) when compared with rats of the same age that fail to surge. However, middle-aged rats with LH surges have significantly lower levels of extracellular Glu in the medial preoptic area (P < 0.05) on the day of the LH surge than young rats (Fig. 5, inset). The LH surge in young rats is associated with 3-fold higher extracellular levels of medial preoptic area Glu (Table 2). Moreover, when a LH surge occurred, mean peak Glu levels in dialysates from middle-aged rats were half as much as peak medial preoptic area Glu levels in young rats (P < 0.01; Table 3). Total medial preoptic area Glu release during the time interval preceding the LH surge was half as much in middle-aged (P < 0.01; Table 2) as in young rats. The 50% reduction in total medial preoptic area Glu release observed on the day of the LH surge correlated with a 50% reduction in total LH release from middle-aged rats (Fig. 5). We did not observe age-related differences in the extracellular levels of glycine (data not shown) or nonneurotransmitter pools of amino acids such as serine (Fig. 6).
Loss in temporal synchrony between the LH surge and maximal medial preoptic area Glu levels in middle-aged rats
There was an age-related difference in the timing of the LH surge relative to the time of peak extracellular Glu levels in the medial preoptic area (Fig. 7). The LH surge was preceded by maximal medial preoptic area Glu in 6/7 (86%) young rats compared with one of eight (12.5%) middle-aged rats (P < 0.01). One young rat reached peak medial preoptic area Glu levels during the LH surge. No young rat attained maximal medial preoptic area extracellular Glu levels after the LH surge. In contrast, most middle-aged rats (63%) attained peak levels of extracellular Glu in the medial preoptic area after the LH surge.
Discussion
This study demonstrates that the delayed onset and reduced amplitude of the LH surge in middle-aged female rats is associated with a decline in extracellular Glu levels in the medial preoptic area. Moreover, these alterations in the E2 and P-induced LH surge of middle-aged rats were associated with a change in the timing of maximal levels of extracellular Glu in the medial preoptic area relative to peak LH as well as a reduction in the total amount of extracellular Glu measured in dialysis samples collected on the day of the LH surge. These studies suggest that the delayed and attenuated LH surge that characterizes middle-aged female rats may result from reduced excitatory drive to GnRH neurons in the medial preoptic area. This finding is consistent with the proposal of Wise et al. (30) that the induction of the LH surge in middle-aged rats correlates with reduced stimulation of hypothalamic medial preoptic area GnRH neurons.
Although the importance of Glu and its respective receptors in the induction of the LH surge is clearly established in normal reproductive cycles of young rats (35, 42, 44, 45, 46, 48, 63, 64, 65, 66, 67), the role of Glu neurotransmission in age-related changes in the induction of the LH surge or the transition into reproductive quiescence is not understood well (20, 26, 51, 52, 68). It has been proposed that abnormal elevations and erratic production of gonadal steroids may predispose middle-aged rats to irregular patterns of gonadotropin release (3). To control for this possibility, we ovariectomized young and middle-aged rats that were still showing normal cycles and then used the same hormonal regimen to prime rats with equivalent doses of E2 and P. Serum levels of E2 and P on the morning of the LH surge were quantified and found to be comparable in young and middle-aged rats. Therefore, it is highly unlikely that the observed abnormalities in gonadotropin release were the result of age-related differences in gonadal steroid production, exposure time, or clearance. The observation that baseline LH levels at the beginning of the experiment were comparable in the two age groups (Table 1) suggests further that E2 negative feedback is not compromised in middle-aged rats.
This study demonstrates that E2- and P-primed female rats that exhibit LH surges have approximately 2-fold higher levels of extracellular Glu in the medial preoptic area than rats that fail to surge, regardless of age. Moreover, medial preoptic area Glu release in young rats that surged was twice as high as those measured in middle-aged rats that surged. This pattern of extracellular medial preoptic area Glu levels was observed more than 12 h after lowering the microdialysis probe. Therefore, the observed differences in dialysate levels of Glu were specifically related to an age-related divergence in the response to gonadal steroid priming and not the result of cellular damage induced by lowering the microdialysis probe. Because medial preoptic area serine levels were equivalent in young and middle-aged rats on the day of the LH surge, age-related changes in extracellular Glu most likely reflect changes in the neurotransmitter pools rather than in global amino acid metabolism. These studies strongly suggest that age-related changes in LH positive feedback are related to altered hypothalamic responsiveness to ovarian steroids and, more specifically, a reduction in excitatory input mediated by the neurotransmitter Glu.
Peak extracellular levels of medial preoptic Glu in young rats generally preceded the onset of the LH surge and began to decline around the time of peak LH release. This was not the case in middle-aged rats; instead, there appeared to be a loss in the temporal synchrony between maximal medial preoptic area extracellular Glu levels and the onset of the LH surge. Maximal extracellular Glu levels in the medial preoptic area of middle-aged rats were variable and frequently failed to precede the LH surge. These data suggest that the age-related delay in steroid-induced LH release was related to a change in the timing of medial preoptic area Glu release and Glu-mediated excitatory neurotransmission.
In rodents, neural signals that regulate the LH surge and maintain estrous cyclicity are critically linked to the generation of circadian rhythms by the suprachiasmatic nuclei (SCN). If the neural signals are delayed by as little as 2 h, the induction of the LH surge is severely delayed (69, 70). Wise et al. (70) hypothesized that reproductive aging results from deterioration of the biological clock and a dyssynchrony in the rhythmicity of a number of key neurotransmitters communicating with GnRH neurons or their afferents. Glutamatergic afferents from the anteroventral periventricular nucleus (71) to the medial preoptic area receive afferent input from the SCN (72, 73). Glutamatergic afferents to the medial preoptic area are also reported to arise in the SCN (72). Age-related changes in the temporal relationship between the onset of the LH surge and the peak medial preoptic area Glu release may be another manifestation of a desynchronized biological clock.
Our experiments do not determine whether Glu acts directly on GnRH neurons or indirectly through interneurons, nor do they assess whether there is an age-related compromise in Glu signal transduction (51, 53). Because Glu receptors are expressed on GnRH neurons and increase GnRH gene expression (74, 75), it is possible that reduced extracellular Glu in the medial preoptic area also results in reduced excitation of GnRH neurons, reduced GnRH production and release, and, as a consequence, reduced LH release. Consistent with the hypothesis that reproductive aging is associated with altered glutamatergic neurotransmission, Miller and Gore (26, 52) recently reported alterations in NMDA receptor subunit stoichiometry in middle-aged female rats. They proposed that the age-related changes in NMDA receptor stoichiometry may confer longer excitatory postsynaptic potentials (26, 52). It is possible that age-related changes in the assembly of the NMDA receptor may represent a compensatory mechanism that allows GnRH neurons to respond to decreased local levels of hypothalamic extracellular Glu preceding the LH surge in middle-aged rats beginning the transition into reproductive senescence.
It is important to acknowledge that other neurotransmitters are important for the induction of LH surges and maintenance of estrous cyclicity. -Amino butyric acid (GABA) and NE have critical roles in the induction and timing of the LH surge (76, 77, 78, 79, 80). In adult female rats, GABA is believed to be responsible for the negative feedback effects of estrogen (81, 82). A decrease in hypothalamic GABA release followed by an increase in Glu release is implicated in the generation of the preovulatory GnRH/LH surge. It is this change in the preovulatory ratio of medial preoptic GABA to Glu release that is thought to result in a net excitation of GnRH neurons and increased GnRH release (47). Thus, it is possible that the reduced levels of medial preoptic area Glu release associated with reproductive aging do not yield an environment that favors maximal GnRH neuronal stimulation and a normal preovulatory GnRH surge. There is also evidence that Glu releases NE from hypothalamic slices, an effect that is inhibited by Glu receptor antagonists or GABA receptor agonists (83). The delayed onset and attenuated release of LH is associated with reduced NE turnover (32) and a reduction in medial preoptic area NE release at the time of the LH surge in middle-aged rats (15). Given these findings, it is logical to hypothesize that reduced glutamatergic neurotransmission may indirectly affect the LH surge by altering NE neurotransmission.
In summary, the present studies report the novel finding that the abnormal LH surge observed in middle-aged rats correlates with attenuated extracellular Glu levels in the medial preoptic area. Our data also suggest that the delayed onset of the LH surge in middle-aged rats could be related to a loss in the temporal synchrony between maximal Glu release in the medial preoptic area of the hypothalamus and the LH surge. These observations strongly support the hypothesis that the aging hypothalamus is less responsive to the positive feedback actions of ovarian steroids and that abnormal medial preoptic area Glu release contributes to age-related aberrations in gonadal steroid-induced gonadotropin release.
Acknowledgments
We are thankful for the technical support provided by Cheryl Shaw and Alice Shu for intracerebral microdialysis and LH assays, Tovadel Adel and Gohar Zeitlin for E2 and P assays, and Cindy Song’s assistance with HPLC determination of Glu levels in microdialysis samples.
Footnotes
This work was supported by National Institutes of Health Grants T32 HD40135, RO1 HD29856 and The Robert Wood Johnson Foundation Harold Amos Faculty Development Grant.
Abbreviations: AUC, Area under the curve; E2, estradiol; GABA, -amino butyric acid; Glu, glutamate; NE, norepinephrine; NMDA, N-methyl-D-aspartic acid; P, progesterone; SCN, suprachiasmatic nuclei.
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Levine Neuroscience Laboratory, Department of Neurology (G.D.Z.), University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medial School, Piscataway, New Jersey 08854
Abstract
Glutamate (Glu) and its receptors are involved in the maturation and maintenance of the neural mechanisms governing the preovulatory LH surge of young, reproductive-aged rodents and nonhuman primates. Little is known about the role of Glu in the delayed onset and reduced peak amplitude of the LH surge that characterizes female rodents during early reproductive senescence. The present study tested the hypothesis that the delayed and attenuated LH surge observed in middle-aged female rats is associated with altered hypothalamic Glu release. We used intracerebral microdialysis in young (3–4 months) and middle-aged (9–11 months) female rats to monitor changes in medial preoptic area Glu release and jugular vein catheters to monitor changes in serum LH levels. All animals were ovariectomized and injected with estradiol and progesterone in doses sufficient to produce a robust LH surge in most (70%) young rats. In both young and middle-aged females that surged, extracellular Glu levels were higher than in those that did not surge. Among animals that surged, the onset of the LH surge was significantly delayed, and the amplitude of the surge was significantly reduced in middle-aged compared with young rats. Middle-aged females also had significantly reduced extracellular Glu levels throughout the day of the LH surge when compared with young females. These data strongly suggest that age-related hypothalamic dysfunction contributes to reproductive aging independent of gonadal failure. We propose that reduced medial preoptic area Glu transmission contributes to reproductive aging by attenuating excitatory input to GnRH neurons.
Introduction
REPRODUCTIVE AGING IN female nonhuman primates and rodents is characterized by alterations in the patterns of LH and follicle-stimulating hormone release (1, 2, 3, 4, 5, 6, 7), especially in the periovulatory period. It has been postulated that the age-related decline in functional ovarian reserve (3, 4, 7, 8, 9) and coincident hypoestrogenic and hypoprogestinemic hormonal milieu trigger secondary changes in gonadotropin release patterns. Together, these events were believed to initiate the transition into reproductive quiescence (3, 5, 9, 10). Although it is clear that ovarian failure ultimately defines human reproductive quiescence (11), a growing number of studies implicate a significant neuroendocrine component to reproductive aging in primate and nonprimate models (4, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21).
In rodents, a delayed onset and attenuated amplitude of the preovulatory LH surge heralds the onset of reproductive senescence (18, 21). This change in LH release begins to occur when rodents are 8–12 months old, an age equivalent to human middle age (21, 22). Changes in rodent gonadotropin release patterns precede other indicators of reproductive senescence, such as irregular estrous cyclicity. These age-related changes in gonadotropin release do not correlate with reduced basal GnRH peptide release (20, 23, 24), reduced capacity for activated GnRH neurons to release GnRH (23, 25), or reduced GnRH neuronal density (26, 27). Moreover, alterations in LH release from middle-aged cycling rats do not result from abnormal pituitary responsiveness to estrogen positive feedback (28, 29) or GnRH peptide (22, 23, 28, 29). Age-related changes in gonadotropin release have, however, been associated with a 50% reduction in GnRH neuronal activation when c-Fos was used as a marker for cellular activation (30) and with reduced mediobasal hypothalamic GnRH release on the day of proestrus (31). Similarly, altered circadian rhythms of norepinephrine (NE) turnover (32) and release (15) and attenuated vasoactive intestinal peptide mRNA (33) levels have been reported in specific hypothalamic nuclei of middle-aged rats.
Maturation of the GnRH neuronal network during the pubertal transition and hypothalamic control of estrous cyclicity are thought to involve changes in the balance between excitatory input from glutamatergic and inhibitory input from GABAergic neuronal processes (34, 35, 36, 37, 38, 39, 40, 41). In mature, steroid-primed female rats, increases in hypothalamic glutamate (Glu) release (42) and Glu receptor activation stimulate GnRH neurons (43) and induce GnRH peptide synthesis and release (44, 45) and, consequently, pituitary LH synthesis and release. The preovulatory LH surge is preceded by an increase in extracellular Glu levels (42, 44, 46, 47, 48) in the hypothalamic medial preoptic area. The Glu receptor agonist, N-methyl-D-aspartic acid (NMDA), stimulates GnRH peptide synthesis and secretion and advances the estradiol (E2) and progesterone (P)-induced LH surge (47, 48). Likewise, the NMDA receptor antagonist, MK801, blocks the E2- and P-induced LH surge (47, 48, 49). Although increases in hypothalamic Glu levels and Glu receptor activation are implicated in the regulation of GnRH neuronal activation and GnRH synthesis and release in young rats, the status of hypothalamic Glu neurotransmission and its possible role in the initiation and maintenance of reproductive senescence has received little attention (50, 51, 52, 53). We therefore tested the hypothesis that age-related changes in hypothalamic Glu release contribute to age-related alterations in LH release. We used intravenous jugular vein catheters for serum collection and intracerebral microdialysis in the hypothalamic medial preoptic area of young and middle-aged rats that were ovariectomized and primed with E2 plus P to study the magnitude of extracellular Glu levels attained on the day of the LH surge and the temporal relationships among the attainment of maximal medial preoptic area Glu levels and the onset and amplitude of the LH surge.
Materials and Methods
Animals
Young (3–4 months) and middle-aged (9–11 months) female Sprague Dawley rats were purchased from Taconic Farms (Germantown, NY). All rats had access to food and water ad libitum and were housed individually and maintained on a 14-h light, 10-h dark cycle with lights off at 2000 h. Only those rats that exhibited at least three normal 4-d estrous cycles, as determined by daily vaginal smears, were used for microdialysis and LH release studies.
Stereotaxic surgery and jugular vein catheterization
Young (n = 14) and middle-aged (n = 16) rats that exhibited normal estrous cycles were anesthetized with im ketamine (80 mg/kg) and xylazine (4 mg/kg) for ovariohysterectomy (54) and intracerebral microdialysis guide cannula placement. Anesthetized rats were placed in a Kopf stereotaxic apparatus and secured with ear bars and a nose piece set at +5 mm. Using Bregma as a landmark and stereotaxic coordinates provided in the atlas of Pellegrino et al. (55) (dorsal/ventral, –8.5; anterior/posterior, +2.0; and medial/lateral, ±0.6), a unilateral 23-gauge guide cannula was implanted in the hypothalamic medial preoptic area. Guide cannulae and concentric dialysis probes (2-mm dialysis surface with 340-μm outer diameter) were purchased from Bioanalytical Systems, Inc. (West Lafayette, IN). All rats were allowed to recover for 7 d. On the 8th postoperative day, rats with intracerebral microdialysis guide cannulae were lightly anesthetized with ketamine/xylazine and an internal jugular vein catheter placed in the right atrium for serial blood sampling (56). Catheters were kept patent with daily infusion of 1 ml heparinized saline (50 IU). All studies were carried out according to protocols approved by the Institutional Animal Care and Use Committee of Albert Einstein College of Medicine and according to National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Gonadal steroid priming
To ensure that young and middle-aged females were exposed to equivalent hormone levels, gonadal steroid priming for the induction of the LH surge was achieved with previously described methods (57). At 0900 h on the day of catheterization, rats were injected sc with 2 μg E2 benzoate (in 0.1 ml peanut oil); a second injection was given 24 h later. To augment the LH surge, a single sc injection of 500 μg P was given at 0900 h, 48 h after the first E2 injection (58). This hormone treatment produced LH surges in eight of 11 young and eight of 12 middle-aged rats.
Microdialysis sampling and serum collection
To avoid acute injury-induced fluctuations in neurotransmitter release at the time of intracerebral microdialysis probe placement (39), probes were lowered at least 12 h before the start of each experiment. Microdialysis sample collection was initiated 1 h before the P injection. The probes were perfused at a rate of 1.25 μl/min with artificial cerebral spinal fluid [124 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 1.8 mM CaCl2, 26 mM NaCO3, and 7.2 mM dextrose (pH 7.4)]. Microdialysis samples were collected from anesthetized, freely moving rats for a total of 13 h at 30-min intervals. All microdialysis samples were acidified to 0.1 N perchloric acid, flash frozen, and stored at –70 C until analysis with HPLC. Blood sampling was initiated at the time of the P injection and continued every 1–2 h for a total of 12 h. Approximately 200 μl blood was collected into Eppendorf tubes containing 100 μl ice-cold heparinized saline (10 IU), refrigerated overnight, and centrifuged at 1000 x g for 10 min. Serum was removed with a glass pipette and stored at –70 C until assayed for LH. At the end of each experiment, animals were killed with halothane, decapitated, and the brain rapidly dissected and frozen in 2-methylbutane. Frozen brains were stored at –70 C until cryostat sectioning for histological assessment of probe placement.
LH assay
Serum LH levels were determined in duplicate with a rat double antibody LH enzyme immunoassay (Biotrak System, Amersham Bioscience Corp., Piscataway, NJ). The lower limit of the assay was 0.1 ng/ml, and the intra- and interassay coefficients of variation were 8.4 and 11.5%, respectively.
E2 and P assays
Sera collected 2 h after the P injection were used to determine serum P and E2 levels by fluoroimmunoassay using the DELFIA (PerkinElmer Life Sciences, Turku, Finland) E2 and P assays. All sample measurements were performed in duplicate, and the mean value was reported. The lower limit for E2 detection was 13.6 pg/ml, and the intra- and interassay coefficients of variance were 6.1 and 5.8%, respectively. The lower limit for P detection was 0.25 ng/ml, and the intra- and interassay coefficients of variance were 4.9 and 7.0%, respectively.
HPLC analysis of Glu
Amino acid content in medial preoptic area microdialysis samples was quantified by methods previously described (59, 60). Briefly, microdialysis samples were adjusted to pH 5 with 2.5 M potassium carbonate. After o-phthalaldehyde derivatization, amino acids were separated by gradient elution and detected with a Beckman 175 fluorometer. Derivatized samples were applied to a C-18 column (0.4- x 2.5-cm reverse phase, Beckman 5-μm Ultrasphere) at a flow rate of 0.75 ml/min. A programed gradient elution was created with dual HPLC pumps and two solvent mixtures [solvent A, 0.1 M sodium acetate (pH 5.9) in 10% methanol; solvent B, 80% methanol]. Amino acid identification and quantification were achieved by comparing peak retention times and heights of known standards (Sigma, St. Louis, MO) to unknown samples. Amino acid content is reported as picomoles per microliter. The lower limit for detection of Glu is 0.05 pmol/μl.
Histological verification of probe placements
Frozen brains were sectioned in the transverse plane with a cryostat. Every third 40-μm section was saved throughout the extent of the dialysis probe track, stained with thionin, and inspected for confirmation of intracerebral microdialysis probe placement in the medial preoptic area. Only those rats in which probe placement was confirmed were included in the data analysis. Three rats from each age group were discarded due to inaccurate probe placement.
LH surge analysis
A LH surge was defined as an increase in serum LH levels equal to or greater than 1.5 times baseline levels for a minimum of two samples. Baseline LH levels were defined as the LH value observed at the time of the P injection. The LH surge onset was considered delayed if it occurred at a time greater than 2 SD values from the mean onset time of young rats included in our studies. Because our objective was to determine whether the delayed onset and reduced peak amplitude of the LH surge in middle-aged rats was associated with age-related differences in medial preoptic area Glu levels, certain analyses only included data from middle-aged rats that exhibited a delayed and attenuated LH surge and young rats with a normal LH surge (defined as a LH surge with an onset and peak amplitude that falls within 2 SD values of mean values for all young rats with surges). Only one of nine middle-aged rats failed to meet our criterion for a delayed onset of the LH surge and was therefore excluded from data analysis. Only one of eight young rats failed to mount a LH surge within 2 SD values of mean values for all young rats with surges. This rat exhibited a LH surge with a delayed onset and reduced amplitude and was therefore excluded from our data analysis.
Statistical analysis
The integrated area under the curve (AUC) for total medial preoptic area Glu levels and total serum LH release was calculated using Pharm/PCS version 4.2 (61). All data are expressed as mean ± SE. Two-way ANOVA was used to evaluate age-related differences between the AUC of serum LH and medial preoptic area Glu levels in rats with and without a LH surge. Differences in mean peak LH release, pre-LH surge Glu AUC, Ser AUC, mean peak Glu release, and the time of the onset of the LH surge relative to the time of P injection were analyzed with Student’s t tests. Two-way ANOVA was used to compare differences in body weight, plasma E2, P, and baseline LH levels in young and middle-aged rats with and without LH surges. P 0.05 was accepted as statistically significant. A Fisher exact test was used to evaluate age-related differences in the time of peak Glu levels relative to the onset of the LH surge.
Results
Middle-aged rats exhibit a delayed onset and attenuated peak amplitude of the LH surge
Baseline LH values at the time of P injection are equivalent in young and middle-aged rats (Table 1). Two hours after the P injection, gonadal steroid levels were also equivalent in young and middle-aged rats despite their differences in body weight (Table 1). Similar percentages of young (72%) and middle-aged (69%) rats with verified probe placements mounted a LH surge with our E2 and P protocol. Most middle-aged rats (eight of nine; 89%) exhibited a delayed onset and attenuated peak amplitude of the LH surge. Most young rats (seven of eight; 88%) mounted a robust LH surge that begins between 4–5 h after P injection. When compared with young rats, middle-aged rats release half as much LH (P < 0.05) on the day of the steroid-induced LH surge (Table 2 and Fig. 1). Similarly, the mean peak amplitude of LH release in individual middle-aged rats is reduced by approximately 60% (P < 0.01) compared with young rats (Table 3 and Fig. 2). When a LH surge failed to occur, serum LH levels in middle-aged and young rats were equivalent (Table 2). When compared with young rats exhibiting a LH surge, the onset of the LH surge in middle-aged rats was delayed by approximately 3 h (P < 0.01) (Table 3 and Fig. 2). These results are in agreement with others (31, 62).
The LH surge is associated with higher extracellular levels of medial preoptic area Glu
Young and middle-aged rats that have a LH surge have 2-fold higher levels of extracellular Glu in the medial preoptic area than animals of the same age that fail to surge (Figs. 3 and 4). Thus, the LH surge in both young and middle-aged rats is associated with higher levels of extracellular Glu in the medial preoptic area (P < 0.05) when compared with rats of the same age that fail to surge. However, middle-aged rats with LH surges have significantly lower levels of extracellular Glu in the medial preoptic area (P < 0.05) on the day of the LH surge than young rats (Fig. 5, inset). The LH surge in young rats is associated with 3-fold higher extracellular levels of medial preoptic area Glu (Table 2). Moreover, when a LH surge occurred, mean peak Glu levels in dialysates from middle-aged rats were half as much as peak medial preoptic area Glu levels in young rats (P < 0.01; Table 3). Total medial preoptic area Glu release during the time interval preceding the LH surge was half as much in middle-aged (P < 0.01; Table 2) as in young rats. The 50% reduction in total medial preoptic area Glu release observed on the day of the LH surge correlated with a 50% reduction in total LH release from middle-aged rats (Fig. 5). We did not observe age-related differences in the extracellular levels of glycine (data not shown) or nonneurotransmitter pools of amino acids such as serine (Fig. 6).
Loss in temporal synchrony between the LH surge and maximal medial preoptic area Glu levels in middle-aged rats
There was an age-related difference in the timing of the LH surge relative to the time of peak extracellular Glu levels in the medial preoptic area (Fig. 7). The LH surge was preceded by maximal medial preoptic area Glu in 6/7 (86%) young rats compared with one of eight (12.5%) middle-aged rats (P < 0.01). One young rat reached peak medial preoptic area Glu levels during the LH surge. No young rat attained maximal medial preoptic area extracellular Glu levels after the LH surge. In contrast, most middle-aged rats (63%) attained peak levels of extracellular Glu in the medial preoptic area after the LH surge.
Discussion
This study demonstrates that the delayed onset and reduced amplitude of the LH surge in middle-aged female rats is associated with a decline in extracellular Glu levels in the medial preoptic area. Moreover, these alterations in the E2 and P-induced LH surge of middle-aged rats were associated with a change in the timing of maximal levels of extracellular Glu in the medial preoptic area relative to peak LH as well as a reduction in the total amount of extracellular Glu measured in dialysis samples collected on the day of the LH surge. These studies suggest that the delayed and attenuated LH surge that characterizes middle-aged female rats may result from reduced excitatory drive to GnRH neurons in the medial preoptic area. This finding is consistent with the proposal of Wise et al. (30) that the induction of the LH surge in middle-aged rats correlates with reduced stimulation of hypothalamic medial preoptic area GnRH neurons.
Although the importance of Glu and its respective receptors in the induction of the LH surge is clearly established in normal reproductive cycles of young rats (35, 42, 44, 45, 46, 48, 63, 64, 65, 66, 67), the role of Glu neurotransmission in age-related changes in the induction of the LH surge or the transition into reproductive quiescence is not understood well (20, 26, 51, 52, 68). It has been proposed that abnormal elevations and erratic production of gonadal steroids may predispose middle-aged rats to irregular patterns of gonadotropin release (3). To control for this possibility, we ovariectomized young and middle-aged rats that were still showing normal cycles and then used the same hormonal regimen to prime rats with equivalent doses of E2 and P. Serum levels of E2 and P on the morning of the LH surge were quantified and found to be comparable in young and middle-aged rats. Therefore, it is highly unlikely that the observed abnormalities in gonadotropin release were the result of age-related differences in gonadal steroid production, exposure time, or clearance. The observation that baseline LH levels at the beginning of the experiment were comparable in the two age groups (Table 1) suggests further that E2 negative feedback is not compromised in middle-aged rats.
This study demonstrates that E2- and P-primed female rats that exhibit LH surges have approximately 2-fold higher levels of extracellular Glu in the medial preoptic area than rats that fail to surge, regardless of age. Moreover, medial preoptic area Glu release in young rats that surged was twice as high as those measured in middle-aged rats that surged. This pattern of extracellular medial preoptic area Glu levels was observed more than 12 h after lowering the microdialysis probe. Therefore, the observed differences in dialysate levels of Glu were specifically related to an age-related divergence in the response to gonadal steroid priming and not the result of cellular damage induced by lowering the microdialysis probe. Because medial preoptic area serine levels were equivalent in young and middle-aged rats on the day of the LH surge, age-related changes in extracellular Glu most likely reflect changes in the neurotransmitter pools rather than in global amino acid metabolism. These studies strongly suggest that age-related changes in LH positive feedback are related to altered hypothalamic responsiveness to ovarian steroids and, more specifically, a reduction in excitatory input mediated by the neurotransmitter Glu.
Peak extracellular levels of medial preoptic Glu in young rats generally preceded the onset of the LH surge and began to decline around the time of peak LH release. This was not the case in middle-aged rats; instead, there appeared to be a loss in the temporal synchrony between maximal medial preoptic area extracellular Glu levels and the onset of the LH surge. Maximal extracellular Glu levels in the medial preoptic area of middle-aged rats were variable and frequently failed to precede the LH surge. These data suggest that the age-related delay in steroid-induced LH release was related to a change in the timing of medial preoptic area Glu release and Glu-mediated excitatory neurotransmission.
In rodents, neural signals that regulate the LH surge and maintain estrous cyclicity are critically linked to the generation of circadian rhythms by the suprachiasmatic nuclei (SCN). If the neural signals are delayed by as little as 2 h, the induction of the LH surge is severely delayed (69, 70). Wise et al. (70) hypothesized that reproductive aging results from deterioration of the biological clock and a dyssynchrony in the rhythmicity of a number of key neurotransmitters communicating with GnRH neurons or their afferents. Glutamatergic afferents from the anteroventral periventricular nucleus (71) to the medial preoptic area receive afferent input from the SCN (72, 73). Glutamatergic afferents to the medial preoptic area are also reported to arise in the SCN (72). Age-related changes in the temporal relationship between the onset of the LH surge and the peak medial preoptic area Glu release may be another manifestation of a desynchronized biological clock.
Our experiments do not determine whether Glu acts directly on GnRH neurons or indirectly through interneurons, nor do they assess whether there is an age-related compromise in Glu signal transduction (51, 53). Because Glu receptors are expressed on GnRH neurons and increase GnRH gene expression (74, 75), it is possible that reduced extracellular Glu in the medial preoptic area also results in reduced excitation of GnRH neurons, reduced GnRH production and release, and, as a consequence, reduced LH release. Consistent with the hypothesis that reproductive aging is associated with altered glutamatergic neurotransmission, Miller and Gore (26, 52) recently reported alterations in NMDA receptor subunit stoichiometry in middle-aged female rats. They proposed that the age-related changes in NMDA receptor stoichiometry may confer longer excitatory postsynaptic potentials (26, 52). It is possible that age-related changes in the assembly of the NMDA receptor may represent a compensatory mechanism that allows GnRH neurons to respond to decreased local levels of hypothalamic extracellular Glu preceding the LH surge in middle-aged rats beginning the transition into reproductive senescence.
It is important to acknowledge that other neurotransmitters are important for the induction of LH surges and maintenance of estrous cyclicity. -Amino butyric acid (GABA) and NE have critical roles in the induction and timing of the LH surge (76, 77, 78, 79, 80). In adult female rats, GABA is believed to be responsible for the negative feedback effects of estrogen (81, 82). A decrease in hypothalamic GABA release followed by an increase in Glu release is implicated in the generation of the preovulatory GnRH/LH surge. It is this change in the preovulatory ratio of medial preoptic GABA to Glu release that is thought to result in a net excitation of GnRH neurons and increased GnRH release (47). Thus, it is possible that the reduced levels of medial preoptic area Glu release associated with reproductive aging do not yield an environment that favors maximal GnRH neuronal stimulation and a normal preovulatory GnRH surge. There is also evidence that Glu releases NE from hypothalamic slices, an effect that is inhibited by Glu receptor antagonists or GABA receptor agonists (83). The delayed onset and attenuated release of LH is associated with reduced NE turnover (32) and a reduction in medial preoptic area NE release at the time of the LH surge in middle-aged rats (15). Given these findings, it is logical to hypothesize that reduced glutamatergic neurotransmission may indirectly affect the LH surge by altering NE neurotransmission.
In summary, the present studies report the novel finding that the abnormal LH surge observed in middle-aged rats correlates with attenuated extracellular Glu levels in the medial preoptic area. Our data also suggest that the delayed onset of the LH surge in middle-aged rats could be related to a loss in the temporal synchrony between maximal Glu release in the medial preoptic area of the hypothalamus and the LH surge. These observations strongly support the hypothesis that the aging hypothalamus is less responsive to the positive feedback actions of ovarian steroids and that abnormal medial preoptic area Glu release contributes to age-related aberrations in gonadal steroid-induced gonadotropin release.
Acknowledgments
We are thankful for the technical support provided by Cheryl Shaw and Alice Shu for intracerebral microdialysis and LH assays, Tovadel Adel and Gohar Zeitlin for E2 and P assays, and Cindy Song’s assistance with HPLC determination of Glu levels in microdialysis samples.
Footnotes
This work was supported by National Institutes of Health Grants T32 HD40135, RO1 HD29856 and The Robert Wood Johnson Foundation Harold Amos Faculty Development Grant.
Abbreviations: AUC, Area under the curve; E2, estradiol; GABA, -amino butyric acid; Glu, glutamate; NE, norepinephrine; NMDA, N-methyl-D-aspartic acid; P, progesterone; SCN, suprachiasmatic nuclei.
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