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Endocrinology Vol. 143, No. 12 4837-4845
Copyright © 2002 by The Endocrine Society

ARTICLE

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

Erich N. Ottem, Jonathan G. Godwin and Sandra L. Petersen

Department of Biology, Neuroscience and Behavior Program, and Center for Neuroendocrine Studies, University of Massachusetts, Amherst, Massachusetts 01003

Address all correspondence and requests for reprints to: Dr. Sandra L. Petersen, Department of Biology, University of Massachusetts, Amherst, Massachusetts 01003. E-mail: sandyp{at}bio.umass.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although estradiol (E2) triggers phasic increases in LH-releasing hormone (LHRH) synthesis and release, the neurocircuitry responsible for these changes is unclear. We used an ovariectomized, E2-treated animal model to investigate the possibility that glutamate, through N-methyl-D-aspartate (NMDA) receptors (NMDAR), communicates E2 signals to LHRH neurons. A neuroanatomical analysis of the region containing LHRH neurons revealed that approximately 80% of LHRH neurons in medial, but less than 40% in lateral, nuclei of the preoptic area contained NMDAR1 mRNA. Consistent with this distribution pattern, NMDA doubled LHRH mRNA levels in medial neurons, but increased them by less than 30% in cells of the lateral nuclei. Steroids did not alter NMDAR1 mRNA levels in LHRH neurons or change the percentage of LHRH neurons expressing the gene. Furthermore, in contrast to the regionalized effects of NMDA, E2 treatment increased LHRH mRNA levels to the same extent in medial and lateral neurons, and MK801 failed to block E2-induced changes in LHRH gene expression. These results demonstrate that glutamatergic signaling via NMDA receptors is direct and preferentially targets LHRH neurons in medial nuclei of the preoptic area. However, it is unlikely that NMDAR activation mediates E2-dependent increases in LHRH mRNA levels before the LH surge.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTRADIOL (E2) stimulates LH-releasing hormone (LHRH) synthesis (1, 2, 3) and release (4) from neurons in the preoptic area (POA). These changes are critical for the preovulatory surge of LH release, but the neural mechanisms responsible are unclear. We showed previously that microimplants of antiestrogen placed into the POA block both E2-dependent increases in LH surge release and LHRH gene expression (5). These findings suggest that the E2 signal responsible for activating LHRH neurons is triggered locally in the POA. Considering that glutamatergic neurons are the major excitatory neuronal type in the POA (6), they are likely candidates for transmitting stimulatory signals to LHRH neurons.

Although activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor stimulates LHRH and LH release (7, 8, 9, 10, 11, 12, 13, 14), the question of whether LHRH neurons contain NMDA receptors has not been resolved. NMDA triggers LHRH synthesis, release, and electrical activity in immortalized LHRH neurons and GT1 cells (15, 16, 17, 18), and NMDA receptor type 1 (NMDAR1) gene expression (but not protein) (19) has been detected in GT1 cells (20). Furthermore, although in vivo studies from the Gore laboratory originally found that only 19% of LHRH neurons in postpubertal (21) and 30% in young proestrous (22) rats contained NMDAR1, they recently revised the estimate, reporting that approximately 54% of LHRH neurons contain NMDAR1 immunoreactivity in young rats on proestrus (13). Similarly, a recent report showed that in GnRH promoter-lacZ transgenic mice, between 40–50% of ß-galactosidase-containing neurons also contain NMDAR1 immunoreactivity on postnatal d 30 (23). Thus, there is convincing evidence that LHRH neurons are targeted directly by glutamate.

Despite this strong evidence for direct regulation of LHRH neurons through NMDA receptors, no laboratory has detected NMDAR1 mRNA in more than a few LHRH neurons in vivo (19, 24, 25) in adult females. This lack of corroborating evidence prompts the suggestion that immunoreactivity detected in LHRH neurons may not be specific for NMDAR1 or that GT1 cells may not be appropriate models for mature neurons. This view is supported by work in hamsters showing that LHRH neurons are resistant to glutamate toxicity induced by NMDA administration (26). These findings are consistent with the idea that glutamate signals reach LHRH neurons through an indirect route.

The advent of transgenic mice models in which LHRH neurons are identified using green fluorescent protein has allowed a more direct investigation of whether LHRH neurons are regulated through NMDA receptors. Unfortunately, these studies have also provided disparate results. One laboratory found that only 20% of identified LHRH neurons in animals, examined between the ages of 1 wk and 6 months, evidenced functional NMDAR (27). In contrast, another group showed that all LHRH neurons of females examined between postnatal d 17 and 25 responded to NMDA with increased firing rates (28). These discrepancies may be attributable to age, sex, or methods used. Regardless of the reason for these differences, the use of electrophysiological approaches has also failed to resolve the issue of whether a significant number of LHRH neurons contain functional NMDAR.

In addition to the controversy over whether a significant number of LHRH neurons contain bona fide NMDA receptors, it is not clear whether glutamate mediates the effects of E2 on LHRH and LH surge release. Several early studies showed that E2 increases LHRH gene expression in ovariectomized (OVX) rats (29, 30, 31). Subsequently, we found that E2 increases LHRH mRNA during the morning hours and that both this morning rise in gene expression and the afternoon LH surge release are blocked by antiestrogen microimplants into the POA (5). These findings suggest that the events that trigger morning and afternoon changes in LHRH neurons are mediated by the same signal. Numerous studies demonstrate that NMDA increases LHRH gene expression (20, 32, 33, 34), and therefore, glutamate release could be the E2-sensitive signal linking LHRH gene transcription and LH surge release.

Although it is possible that NMDA receptor activation is responsible for E2-induced LHRH gene expression linked to LH surge release, some evidence argues against this idea. In previous work we showed that E2-dependent LHRH gene expression is mediated by transcriptional mechanisms (35), and other investigators found that NMDA activation of LHRH gene expression is not (21, 36). However, in our studies we demonstrated that E2 induces LHRH gene expression preferentially in a rostral population of LHRH neurons residing in the region around the organum vasculosum of the lamina terminalis (OVLT) (3, 35). In contrast, studies examining the effects of NMDA on LHRH gene transcription pooled LHRH neurons from the entire POA (21, 36). Consequently, if only a subpopulation of LHRH neurons contains NMDA receptors, it is possible that direct activation of LHRH gene transcription in this subpopulation would be masked.

Therefore, to reevaluate the hypothesis that glutamatergic neurons directly communicate E2 signals to LHRH neurons through NMDA receptors, we performed several studies. First, we conducted a detailed regional analysis of NMDAR1 and LHRH mRNA colocalization in the POA, simultaneously evaluating the effects of steroids on the incidence of colocalization and on levels of NDMDAR1 mRNA in LHRH neurons. Second, we administered NMDA and performed a similar regional analysis to verify that NMDA receptors found in subpopulations of LHRH neurons were functional. Finally, we examined E2-dependent increases in LHRH mRNA to determine whether they occurred preferentially in subpopulations of neurons that expressed the NMDAR1 gene and whether the NMDA receptor antagonist, MK801, blocked them.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal and tissue preparation
All animals used in this study were maintained in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals, and the institutional animal care and use committee of University of Massachusetts approved all treatment protocols used. Adult female Sprague Dawley rats (Zivic Miller, Zelienople, PA), weighing approximately 225–275 g, were housed in a temperature- and light-controlled room (14-h light, 10-h dark cycle; lights on at 0500 h) with food and water provided ad libitum.

Study 1: do LHRH neurons in specific subnuclei contain NMDAR1 mRNA, and do steroids affect NMDAR1 gene expression in LHRH neurons?
In this study we used dual-label in situ hybridization histochemistry (ISHH) and a well characterized animal model to determine 1) the percentage of LHRH neurons that contain NMDAR1 mRNA and 2) the neuroanatomical location of cells in which LHRH and NMDAR1 mRNAs were colocalized, and 3) the effects of steroids on NMDAR1 gene expression in LHRH neurons. Animals were bilaterally OVX under methoxyflurane (Schering Plough, Omaha, NE) anesthesia. We then used a well characterized animal model in which E2 levels are sustained at physiological levels for 2 d, a treatment that reliably triggers afternoon surge release in OVX animals (37). One week after OVX (d 0), rats were implanted sc with two SILASTIC brand capsules (Dow Corning Corp., Midland, MI) containing either sesame oil (n = 6) or E2 (3-cm capsules containing 200 µg/ml E2 in sesame oil; n = 13). On d 2, seven of the OVX, E2-treated animals were injected sc with progesterone (P4; 5 mg in sesame oil) at 1000 h. On d 2, all animals were killed between 1530–1600 h, and the brains rapidly removed, frozen on dry ice, wrapped in Parafilm (American Can Co., Neenah, WI), and stored at -80 C in conical tubes.

Study 2: does activation of NMDA receptors increase LHRH gene expression preferentially in subpopulations of neurons that contain NMDAR1 mRNA?
For this study, animals were OVX and implanted with sc capsules of E2 on d 0 as described above. On d 1, a polyethylene cannula was inserted into the right atrium of each animal through the jugular vein. On d 2, rats were infused with NMDA (14 mg/kg, iv; n = 6) or saline vehicle (n = 5) at 0700 h (before the onset of E2-triggered rises in LHRH mRNA) (3). NMDA-treated animals and saline-treated control animals were decapitated 2 h after infusion. The brains were rapidly removed, frozen on dry ice, wrapped in Parafilm, and stored in sealed conical tubes at -80 C.

Study 3: are E2-induced changes in LHRH gene expression mediated by activation of NMDAR?
In this study we used single-label ISHH to examine E2-induced temporal changes in LHRH gene expression in subpopulations of neurons in animals killed at either 0600 or 1200 h on d 2. In addition, we examined the effects of MK801 (Sigma, St. Louis, MO), a noncompetitive NMDAR antagonist, on E2-induced changes in LHRH mRNA levels in medial and lateral nuclei of the POA. For this study, animals were OVX and implanted with sc capsules of E2 on d 0 as described above. On d 2, animals were injected and killed by decapitation as described above at 0600 h (n = 6; before the onset of E2-triggered rises in LHRH mRNA) (3). Other groups of animals received sc injections of either saline (n = 6) or MK801 (0.2 mg/kg; n = 6) at 0900 h and a second injection of vehicle or 0.1 mg/kg MK801 at 1100 h. This dose of MK801 reliably inhibits LH surge release in OVX, steroid-treated rats and in proestrous rats (38, 39, 40, 41, 42, 43). These groups were killed at 1200 h (after LHRH mRNA levels rise in response to E2 in this animal model) (3). Brains were rapidly removed, frozen on dry ice, wrapped in Parafilm, and stored in sealed conical tubes at -80 C.

For all studies, 12-µm cryosections were obtained through the POA (0.1 to -0.26 from bregma) (44). Sections were mounted on gelatin-coated slides and stored at -80 C until ISHH was performed.

Preparation of cRNA probes
To colocalize mRNAs encoding NMDAR1 and LHRH in study 1, we simultaneously hybridized tissues to 33P-labeled cRNA probes for NMDAR1 mRNA and digoxigenin-labeled probes for LHRH mRNA. To determine the effects of NMDA on LHRH gene expression (study 2), we used 35S-labeled cRNA probes to detect LHRH mRNA. The cDNA transcription template for NMDAR1 probe was generated using RT-PCR and RNA obtained from the rat brain using RNA STAT-60 (Tel-Test, Friendswood, TX) using methods described previously (45). The sequence of the PCR 3'-primer was 5'-GTGACGGCTCTGCTGATGGA-3', and that of the 5'-primer was 5'-TCCCATCACTCATTGTGGGC-3'. These primers generated a 646-bp fragment corresponding to bases 875-1520 of the rat NMDAR1 cDNA (46), which includes a region targeted in previous ISHH studies (47). The cDNA fragment was cloned into a pCRII-TOPO cloning vector (Invitrogen, Carlsbad, CA), and the identity of the NMDAR1 sequence ligated into the vector was verified by sequence analysis performed in the Morrill Science Center DNA Sequencing Facility at the University of Massachusetts. The plasmid was linearized with EcoRV for transcription of antisense cRNA probes and with BamHI for sense strand probes.

The template used to prepare cRNA probes for LHRH mRNA was a 330-bp BamHI-HindIII cDNA fragment corresponding to exons I–IV of the LHRH cDNA. Verification of probe specificity has been described previously (48). The template was linearized for transcription of antisense cRNA probes using HindIII.

Radiolabeled probes for NMDAR1 mRNA in study 1 and for LHRH mRNA in study 2 were prepared by in vitro transcription as described previously (3). Ninety picomoles of [33P]UTP (study 1; Perkin-Elmer, Boston, MA) or [35S]UTP (study 2; Perkin-Elmer) were vacuum-dried in a 100-µl microcentrifuge tube, then transcription buffer (Promega Corp., Madison, WI), 10 mM dithiothreitol (DTT; Sigma), 1 µg linearized template, 20 U RNasin (Promega Corp.), 500 µM ATP, 500 µM GTP, 500 µM CTP, 3 µM UTP (Promega Corp.), and 10 U RNA polymerase (Promega Corp.) were added. The mixture was incubated for 30 min at 37 C, then a second aliquot of RNA polymerase was added, and the mixture was incubated for an additional 30 min at 37 C. After the second incubation, the reaction was brought up to 100 µl with nuclease-free water, and the DNA template was digested with 2 U deoxyribonuclease I (Promega Corp.) in the presence of 20 U RNasin, 5 mM Tris-HCl (pH 8.0), 1 mM MgCl2, and 12.5 µg tRNA. The probe was purified by phenol/chloroform extraction, precipitated twice with NaCl and ethanol, and resuspended in 100 µl 10 mM Tris (pH 8.0) and 1 mM EDTA solution.

To colocalize LHRH and NMDAR1 mRNAs in study 1, we transcribed digoxigenin-labeled cRNA probes for LHRH mRNA using 1 µg linearized cDNA template, 20 U T7 polymerase (Promega Corp.), transcription buffer, 500 µM ATP, 500 µM CTP, 500 µM GTP, 50 µM UTP, 250 µM digoxigenin-UTP (Roche, Indianapolis, IN), 10 µM DTT, and 20 U RNasin as described previously (49). This mixture was incubated for 1 h at 37 C, then an additional aliquot of 20 U T7 polymerase was added, and the mixture was incubated for another h at 37 C. The reaction was brought to 100 µl with nuclease-free water, and the DNA template was digested with deoxyribonuclease I (2 U) in the presence of 20 U RNasin. The probe was precipitated twice with NaCl and ethanol, and resuspended in a solution of 50 µl 10 mM Tris, pH 8.0, and 1 mM EDTA.

Dual-label ISHH procedures
In study 1, to verify colocalization of LHRH and NMDAR1 mRNAs, we performed two replicate dual-label ISHH runs using procedures that have been described previously with minor modifications (49). Five to seven tissue sections from every animal in the three treatment groups (OVX, OVX and E2, and OVX, E2, and P4) were included in each ISHH run. Sections were thawed for 10 min, then fixed with 4% formalin in PBS for 15 min and treated with 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl (pH 8.0). Next, tissues were dehydrated in a series of ethanol washes, delipidated in chloroform, then rehydrated in 95% ethanol and allowed to dry. 33P-Labeled NMDAR1 probe (1 x 106 cpm) and digoxigenin-labeled LHRH probe (0.5 µl) were applied to each tissue section in 25 µl hybridization buffer. The hybridization buffer contained 2x standard saline citrate solution (SSC; 1x SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.2), 50% formamide (vol/vol), 10% (wt/vol) dextran sulfate, 250 µg/µl tRNA, 1x Denhardt’s solution (0.02% Ficoll, 0.02% polyvinylpyrrolidine, and 0.02% BSA), and 400 mM DTT. Tissue sections were covered with glass coverslips and incubated at 55 C in humid conditions overnight. After incubation, sections were washed twice for 15 min each time in 1x SSC on an orbital shaker. The sections were then washed twice for 20 min each time in 50% (vol/vol) formamide/2x SSC at 52 C with shaking, followed by two more washes in 2x SSC for 10 min each wash. Sections were then placed in ribonuclease (RNase) buffer [0.5 M NaCl, 10 mM Tris (pH 8.0), and 1 mM EDTA (pH 8.0)] containing 25 µg/ml RNase A (Roche) and incubated at 37 C for 30 min with shaking. The RNase wash was followed by two rinses in 2x SSC for 10 min each time and a final rinse in 50% formamide/2x SSC for 20 min at 52 C. Sections were then processed immediately for immunocytochemical detection of the digoxigenin-labeled probe for LHRH mRNA.

Tissue sections were placed in 3% milk protein (ICN Biomedicals, Inc., Aurora, OH) in TNT buffer (0.1 M Tris-HCl, 0.15 M NaCl, and 0.05% Triton X; Sigma) for 1 h to block nonspecific binding, then washed twice for 5 min in TN buffer (0.1 M Tris-HCl and 0.15 M NaCl). The slides were then incubated at 4 C for 48 h in 2% milk protein in TN buffer containing 1:200 antidigoxigenin conjugated to horseradish peroxidase (Roche), then washed twice for 5 min each time in TN buffer and once in TNT buffer for 5 min. The sections were next incubated for 10 min at room temperature in biotinylated tyramide from a Renaissance TSA-indirect ISH Kit (Perkin-Elmer) in TNT buffer. Next, sections were washed in TNT buffer and incubated at 37 C for 30 min in ABC Elite reagent (Vector Laboratories, Inc., Burlingame, CA), followed by another wash in TNT buffer. Digoxigenin-labeled probe was visualized using TNT buffer containing 10 mg 3,3'-diaminobenzidine tetrahydrochloride (Sigma) and 8 µl 3% hydrogen peroxide. The sections were then rinsed in 0.1 M Tris (pH 8) for 5 min, quickly rinsed in H2O, and rinsed in 70% ethanol for 3 min.

To visualize radiolabeled probe for NMDAR1 mRNA, sections were dipped in NTB3 emulsion (Eastman Kodak Co., Rochester, NY; diluted 1:1 with deionized distilled water) and exposed for 72 h. After the exposure period, the slides were developed in Dektol (Kodak) and fixed in Kodak fixer.

Single-label ISHH
To determine the effects of NMDA on LHRH mRNA levels in study 2 and the effects of E2 and MK801 on these levels in study 3, we used separate single-label ISHH protocols as described previously (50). Fourteen tissue sections from each animal were used and hybridized in a single run per study. Preparation of 35S-labeled cRNA probe to LHRH mRNA, as well as prehybridization, hybridization, and posthybridization procedures were performed as described above with some modifications. 35S-Labeled cRNA probe for LHRH (1 x 106 cpm) was applied to each tissue section in 25 µl hybridization buffer and incubated at 55 C overnight. Posthybridization washes were performed as described in study 1, except that after the final wash in 50% formamide/2x SSC tissue sections were quickly rinsed in distilled, deionized H2O and subjected to a series of washes in ethanol before being allowed to dry. Sections were then dipped in NTB3 emulsion (Kodak) and exposed for 48 h in study 2 and for 24 h in study 3.

Data analysis
In work colocalizing LHRH and NMDAR1 mRNAs (study 1), results of the two hybridization runs were first analyzed separately. Each section was examined under a Leitz Laborlux microscope (Wetzlar, Germany) equipped with a x40 objective to determine the number of LHRH neurons that did or did not contain NMDAR1 mRNA in each of several nuclei of the POA. The assignment of cells was based on a neuroanatomical atlas that details the structure of the POA (44). Our preliminary results indicated that most cells containing both LHRH and NMDAR1 mRNA were located in the more medial nuclei. Therefore, we performed a more formal analysis by comparing colocalization percentages in nuclei defined as medial compared with those classified as lateral based on their proximity to the third ventricle (see Fig. 1Go). The medial group included the median preoptic area and the region of the OVLT as well as the medial septum that, although not part of the POA, also contained LHRH neurons. The lateral group included the medial preoptic area, anteroventral preoptic area, anterodorsal preoptic area, and lateral preoptic area as well as the lateral portions of the nucleus of the diagonal band. Throughout this report, the LHRH cell bodies found in either of the two nuclei groupings will be referred to as the medial group or lateral group. There were no differences between the results of the two hybridization runs, so data were combined for further analysis. Data were analyzed using two-way ANOVA to determine the effects of steroids and neuroanatomical location on the percentage of LHRH neurons that expressed the NMDAR1 gene.



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  		Figure 1. Rostral to caudal arrangements of diagrams detailing the divisions of the POA (44 ). The diagrams represent sections 0.10 mm anterior to bregma (A), at bregma (B), and 0.11 (C) or 0.26 (D) mm posterior to bregma. Labeled regions denote areas in which LHRH mRNA-containing cells were found.

 
In study 1 we also examined all sections included in the second hybridization run to determine steroid effects on levels of NMDAR1 mRNA in medial and lateral groups of LHRH neurons. For this analysis, we used a BioQuant Windows image analysis system (R and M Biometrics, Nashville, TN) equipped with a color video camera (DAGE-MTI, Michigan City, IN) attached to a Laborlux microscope (Leitz, Rockleigh, NJ). We first set a threshold that digitally highlighted pixels corresponding to silver grains (autoradiographic signal for NMDAR1 mRNA). We then determined the number of highlighted pixels overlying brown-colored cells containing digoxigenin-labeled probes to LHRH mRNA. LHRH mRNA-containing neurons were considered to be positive for NMDAR1 gene expression if the number of silver grains over the neuron was at least 5 times the background. Dual-labeled cells were neuroanatomically classified as medial or lateral and assigned a steroid treatment code number with the observer blind to the code. The mean number of highlighted pixels per LHRH neuron was determined for the medial and lateral groups of nuclei in each animal and expressed as the area covered by grains. These means were used to determine grand means for each treatment group. Finally, two-way ANOVA was used to determine the effects of steroids and neuroanatomical location on NMDAR1 gene expression in LHRH neurons.

We analyzed the results of single-label ISHH for LHRH mRNA in studies 2 and 3 as described previously (3) by first setting a threshold to highlight pixels representing silver grains over specifically labeled neurons (those with signal at least 5 times background). The computer then determined the number of highlighted pixels over each cell, and results are expressed as the area covered by pixels. A mean value was obtained for neurons in medial and lateral groups of nuclei of the POA for each animal. These means were used to determine the grand mean for each treatment group and each subdivision. Data were analyzed using two-way ANOVA. Interactions between main effects (treatment and region) were further analyzed with Bonferroni’s t tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Study 1: do LHRH neurons in specific subnuclei contain NMDAR1 mRNA, and do steroids affect NMDAR1 gene expression in LHRH neurons?
A summary of results of dual-label ISHH performed in study 1 is presented in Table 1Go, and examples of LHRH neurons with and without NMDAR1 mRNA are shown in Fig. 2Go. More than twice as many LHRH neurons were detected in the group of nuclei classified as lateral (n = 2622 ± 11.5) than in the medial group of POA nuclei (n = 1279 ± 9.0). There were no significant differences in percentage of colocalization among the various nuclei classified as medial, nor did the incidence of colocalization differ among those in the lateral group of nuclei. However, significantly more LHRH neurons in the medial nuclei expressed the NMDAR1 gene than did those in the lateral nuclei (Table 1Go and Fig. 3Go). Steroids did not affect either the incidence of colocalization (Fig. 3Go) or the mean cellular levels of NMDAR1 mRNA in LHRH neurons in either the medial or lateral group of nuclei (Fig. 4Go).


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  		Table 1. Number of neurons (mean ± SEM) expressing LHRH mRNA or both LHRH and NMDAR1 mRNAs in specific POA nuclei of 1-wk OVX animals receiving various steroid treatments

 


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  		Figure 2. Photomicrographs showing results of dual-label ISHH using 33P-labeled cRNA probes for NMDAR1 mRNA (black silver grains) and digoxigenin-labeled cRNA probes for LHRH mRNA (brown stain). A–C, Representative examples of NMDAR1 mRNA-expressing LHRH neurons; D and E, examples of LHRH neurons that do not contain NMDAR1 mRNA. Scale bar, 1 µm.

 


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  		Figure 3. Histogram showing results of dual-label ISHH studies investigating effects of steroids on the percentage of LHRH neurons that contained NMDAR1 mRNA in medial and lateral groups of POA nuclei. Animals were OVX, implanted sc with SILASTIC brand capsules of E2 or oil on d 0 (1 wk after OVX), and then injected sc with P4 at 1000 h on d 1. Brain tissue was collected between 1530 and 1600 h on d 2. ***, Significantly lower percentage of colocalization than in medial groups of nuclei of same steroid treatment group (P < 0.001).

 


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  		Figure 4. Histogram showing results of dual-label ISHH studies examining effects of steroids on mean cellular levels of NMDAR1 mRNA in LHRH neurons found in medial and lateral groups of POA nuclei. Animals were OVX, implanted sc with SILASTIC brand capsules of E2 or oil on d 0 (1 wk after OVX), and then injected sc with P4 at 1000 h on d 1. Brain tissue was collected between 1530 and 1600 h on d 2.

 
Study 2: does activation of NMDA receptors increase LHRH gene expression preferentially in subpopulations of neurons that contain NMDAR1 mRNA?
In study 2, we found that basal levels of LHRH mRNA were significantly higher in the medial than in the lateral group of nuclei (Fig. 5Go). NMDA significantly increased LHRH mRNA levels in both the medial and lateral groups of nuclei; however, the magnitude of increase in the medial group was nearly 3-fold greater than that in the lateral group (Fig. 5Go).



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  		Figure 5. Histogram showing region-specific effects of NMDA (14 mg/kg, iv) on mean cellular levels of LHRH mRNA in medial and lateral groups of POA nuclei. Animals were OVX, implanted sc with SILASTIC brand capsules of E2 on d 0 (1 wk after OVX), and then infused with NMDA or saline at 0700 h on d 2. Brain tissue was collected for ISHH 2 h after infusions. ***, P < 0.001; *, P < 0.05 (vs. saline treated-animals).

 
Study 3: are E2-induced changes in LHRH gene expression mediated by activation of NMDA receptors?
Results of the single-label ISHH in study 3 are shown in Fig. 6Go. Mean cellular levels of LHRH mRNA levels rose significantly between 0600 and 1200 h in E2-treated animals (Fig. 6Go), but the percent increase did not differ between medial and lateral subpopulations of neurons. Likewise, MK801 treatment did not block the increase in LHRH mRNA levels seen between morning and noon in either the medial or lateral group of nuclei.



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  		Figure 6. Histogram showing effects of E2 and MK801 on mean cellular LHRH mRNA levels in medal and lateral groups of nuclei in the POA. Animals were OVX and implanted sc with capsules of E2 on d 0 (1 wk after OVX). On d 2, one group of animals was killed at 0600 h. Of the remaining groups, one received saline injections, and the other received injections of MK801 (0.2 mg/kg, sc) at 0900 and again at 1100 h (0.1 mg/kg, sc). The latter two groups were killed at 1200 h. **, P < 0.01; *, P < 0.05 (vs. animals killed at 0600 h).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of these studies show that the majority of LHRH neurons located in medial, but not lateral, nuclei of the POA express the NMDAR1 gene. Neither the extent of colocalization nor the level of NMDAR1 mRNA in LHRH neurons was affected by E2 or combined E2 and P4 treatment. Importantly, NMDA increased LHRH gene expression preferentially in the medial subgroup where most neurons expressed the NMDAR1 gene, suggesting that the NMDAR1 mRNA is translated into functional protein. In contrast, there was no regional specificity in the induction of LHRH gene expression by E2, nor did MK801 block the induction. These findings indicate that although glutamatergic signals can be received directly by a medial subpopulation of LHRH neurons containing NMDA receptors, such signaling does not mediate E2-dependent changes in LHRH gene expression.

These studies are the first to detect NMDAR1 mRNA in LHRH neurons and to show that a medial subpopulation of LHRH neurons expresses the NMDAR1 gene. However, previous work showed that only neurons in the rPOA and in the region of the OVLT responded to NMDA with an increase in LHRH gene expression (33). This finding is relevant to the present work because most LHRH neurons in the rPOA/OVLT region are located in nuclei classified as medial in the present studies. Therefore, together these results indicate that the medial subpopulations of LHRH neurons are preferentially regulated by glutamate through the NMDA subtype receptor.

In the present studies we found that unlike the region-specific effects of NMDA, E2 stimulated the same magnitude of increase in LHRH mRNA levels in medial and lateral nuclei. Furthermore, the noncompetitive NMDA receptor antagonist, MK801, did not block E2-induced increases in LHRH gene expression. In this study we examined animals at 0600 and 1200 h, when LH release is still basal in this model (3, 37), so we have no independent evidence that MK801 was effective in blocking NMDA receptor activity in LHRH neurons. However, numerous studies have demonstrated that at the doses used, this drug reliably blocks the LH surge release in OVX, steroid-treated (38, 39, 43) and in proestrous (41, 42) animals. Therefore, it is likely that NMDA receptor activity in LHRH neurons was blocked in our present work. Thus, although abundant evidence shows that NMDA increases LHRH gene expression (22, 32, 33, 34), our present results indicate that glutamatergic signals are not solely responsible for the E2 induction of LHRH gene expression seen before LH surge release. This interpretation is consistent with previous results showing that these increases are triggered by amplified gene transcription (35), an event that is not stimulated by NMDA administration (36).

Our finding that E2 triggered the same response in LHRH gene expression in medial and lateral populations of neurons suggests that NMDA is not responsible for these changes. However, increased gene expression is only one change in LHRH neuronal physiology linked to LH surge release, and therefore, it is possible that NMDA receptor activation may mediate other aspects of E2 signaling to LHRH neurons. Consistent with this idea, previous work demonstrated that medial and lateral subpopulations of LHRH neurons are functionally distinct in biosynthetic responses to steroids. Using computerized three-dimensional reconstruction methods, Hiatt et al. (51) and Rubin et al. (52) showed that levels of LHRH processing increase preferentially in a medially located core population of neurons between diestrus and proestrus. Therefore, although NMDA does not seem to mediate E2 effects on LHRH gene expression, it is possible that glutamatergic signaling plays a role in steroid-induced changes in LHRH processing in the medial subpopulation of neurons.

Evidence suggests that NMDA stimulates LHRH and LH release only in the presence of steroids (53, 54). Consistent with this idea, postpubertal rats have a higher incidence of NMDAR1-immunopositive LHRH neurons than do prepubertal animals (21). However, based on our present results, it does not seem likely that these effects are mediated by steroid regulation of NMDAR1 mRNA levels in LHRH neurons. Neither E2 nor combined E2 and P4 altered the number of LHRH neurons with detectable NMDAR1 mRNA or the level of NMDARI gene expression in LHRH neurons. In agreement with our findings, Gore et al. (22) found that middle-aged animals showing persistent estrus have significantly higher percentages of NMDAR1-expressing LHRH neurons than do middle-aged proestrous animals despite having similar E2 levels. These results, however, do not rule out the possibility that E2 increases levels of functional NMDA receptors in LHRH neurons. Recent evidence indicates that E2 increases the expression of the NMDAR2D subunit gene (55), and this subunit forms heterodimers with NMDAR1 (56). Further studies will be necessary to determine whether the NMDAR2D gene is expressed in the same subpopulation of LHRH neurons as the NMDAR1 gene and whether it is regulated by estrogen in these cells.

Our finding that a significant number of LHRH neurons expressed the NMDAR1 gene differs from the results of previous dual-label ISHH studies in which investigators detected NMDAR1 gene expression in only 5% (24) to 8% (25) of LHRH neurons in rats and none in hamsters (20, 26). It is possible that our ability to detect NMDAR1 mRNA in approximately 50% of LHRH neurons overall while other investigators did not is attributable to the methodologies used. For example, we used shorter probes than those used in previous investigations (24), so the accessibility of the probe to target may have been increased in our studies. In addition, we used 33P rather than 35S to prepare probes, thereby increasing the specific activity of the transcripts and enhancing the sensitivity of the assay. The possibility that these methodological improvements contributed to differences among studies seems likely, because we were able to detect high levels of NMDAR1 mRNA in LHRH neurons after 72 h, whereas previous investigators exposed slides for 1 wk (24) or for 3–8 wk (25).

A second possible explanation for the differences among results of previous studies and our present results is that the regions of the POA chosen for examination may have differed. Most medially located neurons (in which we found the highest percentage of colocalization of LHRH and NMDAR1 mRNAs) reside in the rostral-most portion of the POA, which includes the OVLT. In contrast, more than twice as many LHRH neurons are in lateral nuclei that are generally found in the more caudal regions of the POA. It is not clear what specific regions were considered in previous studies, but a focus on more caudal than rostral regions would markedly affect estimates of NMDAR1 and LHRH colocalization. This issue is particularly important because previous studies colocalizing LHRH and NMDA mRNAs or proteins examined significantly fewer neurons per animal than we did in the present studies. By examining over 250 LHRH neurons per animal and representing all regions of the POA equally, it is possible that we increased the likelihood of finding a subpopulation of LHRH neurons that contain NMDAR1 mRNA.

In summary, we found that most LHRH neurons located in the medial nuclei of the POA express mRNA encoding the NMDAR1 subunit. We further demonstrated that this same subgroup preferentially responds to NMDA with a 2- to 3-fold increase in LHRH mRNA levels, suggesting that the NMDAR1 mRNA is translated into functional protein. Finally, we showed that unlike the effects of NMDA, E2-induced elevations in LHRH gene expression are similar in medial and lateral subpopulations of neurons and are not blocked by MK801. Therefore, although our results suggest that LHRH release activated by NMDA is stimulated directly by glutamate through NMDA receptors, they argue against the concept that E2 induction of LHRH gene expression is mediated solely by glutamatergic signaling to LHRH neurons through NMDA receptors.


   Acknowledgments
 


   Footnotes
 
This work was supported by NIH Grant HD-27305 (to S.L.P.).

Abbreviations: DTT, Dithiothreitol; E2, estradiol; ISHH, in situ hybridization histochemistry; LHRH, LH-releasing hormone; NMDA, N-methyl-D-aspartate; NMDAR1, N-methyl-D-aspartate receptor type 1; OVLT, organum vasculosum of the lamina terminalis; OVX, ovariectomized; P4, progesterone; POA, preoptic area; RNase, ribonuclease; SSC, standard saline citrate solution.

Received July 11, 2002.

Accepted for publication August 26, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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