help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Adams, M. M.
Right arrow Articles by Gore, A. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Adams, M. M.
Right arrow Articles by Gore, A. C.
Endocrinology Vol. 140, No. 5 2288-2296
Copyright © 1999 by The Endocrine Society

ARTICLES

Perinatal Changes in Hypothalamic N-Methyl-D-Aspartate Receptors and Their Relationship to Gonadotropin-Releasing Hormone Neurons1

Michelle M. Adams, Roxana A. Flagg and Andrea C. Gore

Neurobiology of Aging Laboratories and Fishberg Research Center for Neurobiology (M.M.A., R.A.F., A.C.G.), and Henry L. Schwartz Department of Geriatrics and Adult Development (A.C.G.), Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Andrea C. Gore, Ph.D., Mount Sinai School of Medicine, Neurobiology of Aging Laboratories, One Gustave L. Levy Place, Box 1639, New York, New York 10029. E-mail: gore{at}msvax.mssm.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During the neonatal period, the brain is subject to profound alterations in neuronal circuitry due to high levels of synaptogenesis and gliogenesis. In neuroendocrine regions such as the preoptic area-anterior hypothalamus (POA-AH), the site of GnRH perikarya, these changes could affect the maturation of GnRH neurons. Because the GnRH system is developmentally regulated by glutamatergic neurons, we hypothesized that changes in the N-methyl-D-aspartate (NMDA) receptor system begin early in postnatal development, before the onset of puberty, thereby playing a role in establishing the appropriate environment for the subsequent maturation of GnRH neurons. To this end, we determined developmental changes in NMDA receptors, alterations in GnRH gene expression, and the regulation of GnRH neurons by the NMDA receptor system in developing male and female rats. In Exp I, NMDA receptor subunit (NR) 1 mRNA levels in the POA-AH were found to increase significantly (~5-fold) from E18 through P10 in both males and females. NR2b mRNA increased significantly between P0 and P5 in both males and females. In contrast, NR2a subunit mRNA, which was in very low abundance in both males and females, increased only in males between P10 and P15. In Exp II we determined that GnRH gene expression changes differentially in developing male and female rats, with increases from P0 to P5 in males, and decreases from P5 to P10 in females. This latter effect in females is attributed to a change in GnRH gene transcription because GnRH primary transcript RNA levels paralleled changes in GnRH mRNA levels. In Exp III, we tested effects of treatment with an NMDA receptor analog on GnRH mRNA levels and found that only P5 and P10 male rats responded to NMDA receptor activation with an increase in GnRH mRNA levels, via a posttranscriptional mechanism. This greater responsiveness of males to NMDA receptor stimulation may be due to differences in the composition and levels of NMDA receptor subunits. Exp IV examined the localization of NR1 in the POA-AH during neonatal development. No GnRH neurons were immunopositive for NR1, indicating that effects of glutamate on GnRH neurons are mediated by interneurons or other glutamate receptor subunits or types. Taken together, these data indicate that glutamatergic inputs to the POA-AH change dramatically during the early postnatal period, before puberty and before the GnRH system is fully responsive to glutamate, consistent with the hypothesis that the maturation of inputs to GnRH neurons, and the establishment of the proper neurotransmitter "milieu" enabling the activation of GnRH neurons, occurs before the onset of puberty.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PULSATILE release of GnRH is necessary for the regulation of reproductive function. This hormone is synthesized in neurons whose perikarya are located in the preoptic area-anterior hypothalamus (POA-AH) of rodent brains, and is released from neuroterminals in the median eminence into the portal circulation leading to the anterior pituitary. There, it acts on the gonadotropes of the anterior pituitary gland to regulate the synthesis and release of LH and FSH. These peptides then move through the general circulation to the gonads, affecting the synthesis and release of steroid hormones.

The GnRH neurosecretory system is essentially mature at birth with respect to cell number and localization (1, 2, 3); however, endogenous GnRH release is low until the onset of puberty (4). Nevertheless, GnRH cells of immature animals are capable of responding to external stimuli such as electrical and neurotransmitter stimulation by increasing release (5, 6, 7, 8, 9). Thus, it is believed that increases in stimulatory inputs or decreases in inhibitory inputs from neurotransmitters, growth factors and steroid hormones, or a combination of these factors are responsible for the maturation of the GnRH system and the subsequent increase in GnRH release, leading to the pubertal process.

One of the important excitatory inputs to GnRH neurons that plays a role in their maturation is the neurotransmitter glutamate (10, 11). Such a role for glutamate, acting via the N-methyl-D-aspartate (NMDA) receptor, is supported by evidence in adults that NMDA stimulates GnRH and LH release (7, 12, 13, 14) and GnRH gene expression (15, 16). With respect to development, it has also been shown that NMDA can induce precocious puberty in immature rats (12, 16, 17). Increases in glutamate during early development may play a role in synaptogenesis and neurite outgrowth (18, 19, 20), although whether such changes occur specifically on GnRH neurons is unknown. Changes in NMDA receptors are observed during development in several extrahypothalamic regions, and such changes may also occur in the hypothalamus, enabling the glutamate system to have a greater influence on GnRH neurons (21, 22, 23). Therefore, the present study was undertaken to examine developmental changes in glutamatergic inputs in the POA-AH mediated via the NMDA receptor, and the role they play in the regulation of GnRH neurons.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Timed-pregnant Sprague Dawley rats were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN), and housed individually in a temperature-controlled room with a 12-h light, 12-h dark cycle (lights on at 0700). Food and water were available ad libitum. For embryonic day 18 (E18) pups, pregnant females were decapitated, and the fetuses removed. Pups were used at postnatal day (P) 0, 5, 10, or 15.

Experimental design
Exp I: changes in NMDA receptor subunits 1 (NR1), 2a (NR2a), and 2b (NR2b) gene expression during the neonatal period. Male and female rats aged E18, P0, P5, P10, and P15 (n = 5–6) were used in this study. The animals were decapitated, and the preoptic area-anterior hypothalamus (POA-AH) dissected as described previously (24, 25), snap-frozen in liquid Freon on dry ice and stored at -80 C. Cytoplasmic RNA was extracted as described below. NR1 gene expression was measured in a 5 µg aliquot, and NR2a and 2b together in another 5 µg aliquot, by RNase protection assay.

Exp II: changes in GnRH gene expression during the neonatal period. Male and female rats aged P0, P5, P10, and P15 (n = 5–7) were used in this study. Animals were killed, and the POA-AH dissected out and cytoplasmic and nuclear RNA extracted. GnRH cytoplasmic mRNA was measured by RNase protection assay in a 20 µg aliquot of the POA-AH. GnRH primary transcript, an index of gene transcription, was measured by RNase protection assay of nuclear RNA fractions of individual POA-AH dissections.

Experiment III: responsiveness of the GnRH system to NMDA stimulation or inhibition. To test the effects of an NMDA receptor analog, N-methyl-D,L-aspartate (NMA; 5 mg/kg sc) or an NMDA receptor antagonist (MK-801; 0.1 mg/kg sc), animals aged P0, 5, 10, and 15 (n = 3–7) were injected with drug or saline vehicle in 0.1 ml volume, and killed 2 h later. All animals were returned to their mother after the injection to minimize stress; pups were removed for no more than 1 min each and were retrieved by their mother upon return. Animals were killed 2 h after injection, and changes in GnRH gene expression (mRNA and primary transcript) were measured using the same methods as in Exp II.

Experiment IV: neuroanatomical distribution of GnRH neurons and NR1 subunit. Three to six rats of each sex, aged P0, P5–6, P9–10, and P14–15, were deeply anesthetized with ketamine (0.05 ml)/xylazine (0.05 ml) and metofane inhalant and perfused transcardially with 1% paraformaldehyde for 1 min, followed by 4% paraformaldehyde for 10 min. The brains were removed from the skull and postfixed for 6 h at 4 C in 4% paraformaldehyde. Sections (40 µm) were cut on a vibratome (Ted Pella, Redding, CA) and stored in PBS with 0.1% sodium azide. For immunocytochemistry, double-label studies of GnRH with NR1 were performed to assess whether or not the GnRH neurons expressed this NMDA receptor subunit. Additionally, single-label studies of NR1 were performed to determine the density and distribution of this receptor in the POA-AH during development.

RNA extraction and RNase protection assay
Cytoplasmic and nuclear RNA were extracted separately from the POA-AH using a double-detergent cushion/lysis buffer system as described previously (16, 24, 25). Briefly, both fractions were treated with proteinase K (200 µg/ml), and the nuclear fraction subjected to DNase I treatment (60 U) before precipitation. Cytoplasmic and nuclear RNA were resuspended in 20 µl of hybridization solution (0.1 M EDTA, pH 8, and 4 M guanidine thiocyanate; final pH 7.5) for RNase protection assay. The following DNA subclones were used as probes: 1) GnRH complementary DNA (cGnRH), 362 bp in length, spanning the HindIII site in exon 1 to the BamHI site in exon 4, and subcloned into a pBS(+) vector (Stratagene, La Jolla, CA) to measure GnRH mRNA in the cytoplasm (26); (2) a proGnRH (B3C) genomic fragment spanning 506 bp of the intron B-exon 3-intron C junction and subcloned in the EcoRI and HindIII sites of a pBS(+) vector to measure GnRH primary transcript in the nucleus (25), an index of GnRH gene transcription (27); (3) cyclophilin (1B15), an internal control that has previously been shown not to be developmentally regulated in neonatal rats (26), was measured using a 111 bp cDNA clone, spanning from the PstI and XmnI restriction sites and subcloned in a Bluescript KS(+) vector (26); (4) NR1 mRNA in the cytoplasm was measured using a cDNA clone complementary to 284 bp of the N terminus, spanning the BamHI and HindIII restriction sites and subcloned into a Bluescript KS(+) vector [kindly provided by Dr. Stuart Sealfon, Mount Sinai Medical Center, New York, NY; (16)]; (5) NR2a and (6) NR2b mRNAs in the cytoplasm were measured using cDNA clones complementary to bases 1585–2154 and 1423–1992, respectively, cloned into the SmaI site of the pBluescript II SK (+) vector [kindly provided by Drs. S. A. Lipton and N. J. Sucher (28, 29)].

Solution hybridization/RNase protection was performed as described previously (24, 25). Briefly, cGnRH, B3C, NR1, NR2a and NR2b probes were labeled with [{alpha}-32P]UTP to high specific activity (~1,300,000 cpm/ng) and 1B15 probe was labeled to low specific activity (~60,000 cpm/ng) in a final volume of 25 µl (20 µl of RNA and 5 µl of probe). Cytoplasmic samples were incubated with cGnRH and 1B15 probes in the same tubes. For standard curves, probes were mixed with increasing known amounts of cGnRH (0–1.25 pg), 1B15 (0–250 pg), B3C (0–0.5 pg), NR1 (0–100 pg), NR2a (0–1 pg) or NR2b (0–1 pg) reference RNAs. Samples (POA-AH RNA) and standards were allowed to hybridize with probe for 16–18 h at 30 C; the remainder of the assay was conducted as described previously (16, 24, 25). Gels were exposed to x-ray film for 18–36 h to produce an autoradiogram and to a phosphor-imaging screen (Molecular Dynamics, Inc., Sunnyvale, CA) for 18 h for quantitation. The amount of radioactivity in each sample was determined by comparison with the amount of reference RNA calculated by regression analysis.

Immunocytochemistry
Immunocytochemistry was performed with the rabbit polyclonal antibody to GnRH [HU60; kindly provided by Dr. Henryk Urbanski (30)] and the mouse monoclonal antibody to NR1 [54.1; kindly provided by Dr. John H. Morrison (31, 32)]. Sections were rinsed for 30 min in PBS and then preincubated in 2% Normal Goat Serum/2% Normal Horse Serum for 1 h. Then they were transferred to primary antibody (NR1: 1:2000 and GnRH: 1:1000) and put at 4 C for 48 h. Next, the sections were rinsed for 30 min in PBS and transferred to secondary antibody (1:200 biotinylated horse-antimouse IgG; 1:200 Texas red goat-antirabbit IgG) for 1.5 h. Then the sections were rinsed for 30 min in PBS, and put in FITC-avidin D (1:200) for 1.5 h, rinsed, and mounted in PBS onto gelatin-subbed slides. The slides were dried overnight and coverslipped with Vectashield (Vector Laboratories, Inc., Burlingame, CA).

Analysis
The amount of GnRH and NR1 mRNA in POA-AH dissections in Exps I–III was normalized to cyclophilin mRNA levels in the same sample to minimize gel-loading variation, as described previously (16, 25, 33). Cyclophilin mRNA levels did not vary by age or gender in the present study (data not shown). Levels of GnRH primary transcript determined by the B3C probe were expressed in fg per POA-AH nuclear fraction. Changes in levels of each RNA transcript were compared across development by ANOVA, followed by Fisher’s protected least significant difference post hoc test. Significance was set at P < 0.05.

The sections processed for GnRH and NR1 immunocytochemistry, from Experiment IV, were examined with a Zeiss Axiophot fluorescence microscope (Carl Zeiss, Germany) equipped with the appropriate filters for visualization of the fluorescent signals. For each animal, six sections from the level of the OVLT/POA were immunostained and evaluated for double-label immunofluorescence. For these studies, every GnRH neuron within the section was identified and counted at 200x. Additionally, the neuron was scored as NR1-positive or NR1-negative. Omission of the primary antibodies was used in control experiments. Subsequent to this data collection, the sections were reexamined at 630x using a Plan-Neophor 63x/1.25 numerical aperture oil objective on a Zeiss LSM 410 inverted confocal microscope.

In the single-label experiments, a qualitative analysis of the distribution of NR1-immunoreactivity was performed on six sections, adjacent to the previous ones, using the Zeiss Axiophot and was confirmed using confocal microscopy. In both cases, a suitable contrast/brightness setting that yielded a high-resolution image for the cells was determined and used to produce the images. The stored images then were transferred to Adobe Photoshop and printed with a Fuijix Pictrography 3000 printer (Prographics, New York, NY).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp I: changes in NMDA receptor subunit gene expression during the neonatal period
NR1. A representative autoradiogram showing developmental changes in NR1 mRNA in individual POA-AH dissections is shown in Fig. 1Go. ANOVA demonstrated a significant change in NR1 mRNA levels with development (P < 0.0001) that was similar in both male and female rats (Fig. 2Go). NR1 mRNA levels increased approximately 5-fold from E18 through P15, and the increases were significantly different between each of the age groups (P < 0.0001) except P10 and P15.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 1. Developmental changes in NR1 mRNA levels in individual POA-AH dissections of male and female rats. A representative composite autoradiogram of an RNase protection assay for NR1 mRNA is shown. A standard curve with increasing amounts of NR1 reference RNA is shown on the left, and individual POA-AH dissections for developing male (top) and female (bottom) rats on the right. Five micrograms of RNA extracted from a POA-AH dissection were loaded in each lane. Levels of NR1 mRNA in the POA-AH were similar for both developing males and females and increased during development.

 


View larger version (12K):
[in this window]
[in a new window]
  		Figure 2. Developmental changes in NR1 mRNA levels in the POA-AH of male (A) and female (B) rats. In both male and female rats, a significant change in NR1 mRNA levels occurred developmentally with a 5-fold increase from E18 through P15 (P < 0.0001). Levels were significantly different between each age group, except P10 and P15 (a, P < 0.0001 vs. all other ages; b, P < 0.0001 vs. E18, P0, and P5).

 
NR2a. NR2a mRNA levels were substantially lower than NR1 mRNA levels and were undectectable at E18 in both males and females (Fig. 3Go). In males, NR2a was first detectable at P0, whereas in females, NR2a was only detectable at P5 and P10. ANOVA indicated a significant effect of sex (P < 0.04) but not age (P = 0.129) and no significant interaction of age and sex (P = 0.087) on NR2a mRNA levels.



View larger version (6K):
[in this window]
[in a new window]
  		Figure 3. Changes in NR2a mRNA in the POA-AH of developing male (A) and female (B) rats. NR2a mRNA was first detected in males at P0, and in females at P5, and remained low except in P15 males (*, P < 0.05 vs. P15 female).

 
NR2b. NR2b mRNA levels were also of lower abundance than NR1 mRNA, but higher than NR2a mRNA. A significant effect of age on NR2b mRNA (P < 0.0001) but not sex (P = 0.488), and no interaction of age with sex (P = 0.068) was observed (Fig. 4Go). With respect to age, NR2b mRNA levels were significant lower at E18 and P0 compared with P5 through P15.



View larger version (10K):
[in this window]
[in a new window]
  		Figure 4. Changes in NR2b mRNA in the POA-AH of developing male (A) and female (B) rats. NR2b mRNA levels increased significantly between P0 and P5 in developing rats (a, P < 0.05 vs. P5 through P15).

 
Exp II: changes in GnRH gene expression during the neonatal period
Overall, GnRH mRNA and primary transcript levels were similar in male and female rats. A representative autoradiogram showing GnRH mRNA in the cytoplasm and GnRH primary transcript in the nucleus is shown in Fig. 5Go. For both sexes, GnRH mRNA levels were developmentally regulated (P < 0.0001; Fig. 6Go). In males, GnRH mRNA levels increased significantly from P0 to P5 (P < 0.05; Fig. 6aGo), and in females, they decreased significantly from P5 to P10 (P < 0.05; Fig. 6bGo). For GnRH primary transcript levels in the nucleus, there was also a significant developmental effect (P < 0.0001; Fig. 7Go). While in male rats GnRH primary transcript levels did not undergo significant developmental changes (Fig. 7aGo), in females, these levels decreased developmentally from P5 to P10 (P < 0.01; Fig. 7bGo) in parallel with GnRH mRNA levels. GnRH primary transcript levels in females then increased significantly from P10 to P15 (P < 0.005; Fig. 7bGo).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 5. Developmental changes in GnRH mRNA levels in the cytoplasm (A), and GnRH primary transcript in the nucleus (B) of individual POA-AH dissections of male and female rats. Representative autoradiograms of RNase protection assays are shown. In (A), 20 µg of cytoplasmic mRNA extracted from a POA-AH dissection were loaded in each lane. In (B), the entire nuclear fraction of an individual POA-AH was loaded into each lane.

 


View larger version (11K):
[in this window]
[in a new window]
  		Figure 6. Developmental changes in GnRH mRNA levels in the POA-AH of male (A) and female (B) rats. A significant effect of development on GnRH mRNA levels was observed in male and female rats (P < 0.0001). GnRH mRNA levels increased significantly from P0 to P5 in males, and decreased significantly in females from P5 to P10 (a, P < 0.05 vs. P0; b, P < 0.05 vs. P5).

 


View larger version (10K):
[in this window]
[in a new window]
  		Figure 7. Developmental changes in GnRH primary transcript levels in the POA-AH of male (A) and female (B) rats. GnRH primary transcript levels were significantly different in developing rats (P < 0.0001). While no developmental changes were observed in males, in females, GnRH primary transcript levels decreased significantly from P5 to P10, in parallel with the changes that were seen in the mRNA levels. An increase in GnRH primary transcript was observed in females from P10 to P15 (aP < 0.01 vs. P5 and P15).

 
Exp III: responsiveness of the GnRH system to NMDA stimulation or inhibition
ANOVA indicated that there was a significant effect of treatment with the glutamate analog, NMA, or the NMDA receptor antagonist, MK-801, on GnRH mRNA levels (P < 0.05; Table 1Go). In male rats, NMA caused a significant stimulation of GnRH mRNA levels at P5 and P10 (P < 0.05; Table 1Go). There was no effect of MK-801 in males, and in females, neither NMA nor MK-801 caused any change in GnRH mRNA levels (Table 1Go). For GnRH primary transcript levels, there was no significant effect of NMA or MK-801 in either male or female rats (Table 1Go).


View this table:
[in this window]
[in a new window]
  		Table 1. Effects of NMA or MK-801 treatment on GnRH mRNA and primary transcript levels in the POA-AH of male and female rats

 
Exp IV: neuroanatomical distribution of GnRH neurons and NR1 subunit
To determine whether effects of NMDA receptor activation on the GnRH system are mediated directly on GnRH neurons, or indirectly via interneurons, double-label immunocytochemistry for NR1 and GnRH was performed. Immunofluorescent GnRH and NR1-positive neurons were found in the entire rostral-caudal extent of the OVLT/POA in all of the 38 rats examined. In the present study, six sections per animal were processed and evaluated for the colocalization of GnRH and NR1, and an adjacent six sections for the changes in distribution and density of NR1-immunoreactivity. A similar number of GnRH neurons were identified at all age groups and between male and female rats, consistent with previous reports (1, 16).

The results of the double-label experiment showed that in both male and female rats no GnRH neurons colocalized with NR1 at any of the different developmental ages from P0 through P15 (Fig. 8aGo). However, qualitative single-label immunocytochemistry for NR1 indicated that the distribution and density of the NR1-immunoreactivity changed developmentally, and with a similar pattern in both males and females (Fig. 8bGo). In P0 rats, the NR1-immunoreactive cells were sparsely distributed through the OVLT/POA. Most labeled cells were observed around the third ventricle and near the ventral surface of the brain. In contrast, in P5–6 rats, the NR1-immunoreactive cells appeared much more densely packed, again especially along the ventricular and ventral areas. More labeled cells were also observed laterally. In P9–10 rats, NR1-immunoreactivity was still high, and cells appeared to have a patchy distribution. Finally, in P14–15 rats, the NR1-immunoreactive cells were distributed more sparsely than in P9–10 rats but were still found in patches. It should be noted that using the NR1 antibody (54.1), background staining in the neuropil varied considerably during development, with the highest background in P0 pups (Fig. 8aGo), possibly due to differences in the number and distribution of glia and neurons during this early developmental period. Such differences precluded quantitative analyses of NR1 immunofluorescence.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 8. Developmental changes in the neuroanatomical distribution of GnRH neurons and the NR1 subunit in the OVLT/POA of male and female rats. A, Representative photomicrographs from each age group are presented to show double-label immunofluorescence for GnRH (top, red) and NR1 (bottom, green). Immunocytochemical results were similar in males and females; the P0 photomicrograph is from a male pup, and the P6, 10, and 14 micrographs are from females. No GnRH neuron was double-labeled with NR1 during this perinatal period. The asterisk shows the nucleus of the cell. B, Changes in the distribution and density of NR1-immunoreactivity during this developmental period are shown. These micrographs were made from different pups than in A, and micrographs are from females at P0, 6 and 10, and from a male at P14. The NR1-immunoreactive cells changed from a sparse distribution at P0, to a more densely packed one by P5–6. This dense distribution then became more patchy at ages P9–10, and by P14–15 the number of cells appeared slightly more sparse but still in patches. V, Third ventricle. In both experiments, there were no differences observed between male and female rats.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study support our hypothesis that developmental alterations in the NMDA receptor system in the POA-AH, the site of GnRH somata, occur neonatally, before the onset of puberty. We found that NR1, NR2a, and NR2b mRNA levels changed significantly and differentially during perinatal development. Such developmental changes in NMDA receptor biosynthesis, if reflected by changes in protein, could have profound consequences on the properties of the NMDA receptor. This could result in altered glutamate binding and signal transduction and may explain differential developmental and gender-related sensitivities to NMDA receptor activation during reproductive development. Thus, the present results suggest that alterations in neurotransmitter inputs in the region of the GnRH neurosecretory system occur early in development, with potential consequences for establishing the proper neuroendocrine milieu to enable the subsequent increase in pulsatile GnRH release that is responsible for the onset of puberty.

In Exp I, we observed that NR1 mRNA levels increased significantly from E18 through P10 in male and female rats. The presence of the NR1 subunit is mandatory for a functional NMDA receptor (28, 34). It has previously been reported that a small but significant increase in NR1 mRNA occurred between P10 and P20 in rats in one study (16) and between P15 and P20 in another study (35), after which the levels of this subunit were maintained from the pubertal period through adulthood (16). The observation in the present study of an approximately 5-fold increase in NR1 from E18 through P10 probably represents the period during which the most dramatic changes in NR1 occur in the POA-AH compared with other periods during the lifespan of the rat (16, 36). Thus, this aspect of the neuroendocrine environment is essentially established by P10, long before the onset of puberty.

It is thought that in vivo, NMDA receptors exist in pentameric conformations consisting of the NR1 subunit as well as other members of the NR2 subunit family (28). Changes in the relative abundance of the different NR2 subunits and their ratio to the NR1 subunit alter the channel properties (28). Developmental changes in NR2 subunits have been reported in other brain regions such as hippocampus, cortex, and cerebellum (23, 37, 38, 39). In the present study we measured NR2a and 2b mRNAs, which are abundant in the POA-AH and which are expressed at higher levels in neuroendocrine regions than the NR2c and 2d subunits (40). We were particularly interested in quantitating NR2a mRNA because it colocalizes in relatively high levels (~17%) in GnRH neurons of adult female rats (40) and is expressed in GnRH neurons of rats as young as P20 (41). In the present study, NR2a mRNA levels were quite low in the POA-AH at this early developmental period, and we first observed this transcript at low levels in P0 males, and P5 females. For the NR2b subunit, we observed a significant increase in NR2b mRNA levels in both males and females between P0 and P5. Thus, if mRNA levels are reasonably reflective of their corresponding proteins, alterations in the ratio of NR1 to NR2a and 2b subunits in the POA-AH could be involved in setting up the proper environment to facilitate neuroendocrine development.

In Exp II, we examined changes in GnRH gene expression during the early postnatal period. Basal GnRH mRNA levels increased from P0 to P5 in male rats, and decreased from P5 to P10 in female rats, similar to a previous report (26). To determine the molecular mechanism for these changes in GnRH mRNA levels, we measured nuclear GnRH primary transcript levels, an index of GnRH gene transcription (27) in these rats in a separate RNase protection assay. GnRH primary transcript levels did not change in male rats, but changed in parallel with GnRH mRNA levels in female rats. Thus, a transcriptional mechanism for the regulation of GnRH mRNA levels appears to be important for the decrease in GnRH mRNA levels from P5 to P10 in female rats, but not for the increase from P0 to P5 in male rats. It is currently unknown why GnRH mRNA levels are differentially regulated in male and female neonatal rats. The first postnatal week of life is a critical period for sexual differentiation in rats (42), and sex differences in GnRH mRNA levels observed in the present study, as well as in gonadotropin levels as measured in other studies (43, 44, 45), may be due to different exposures to neonatal steroid hormones. It is important to note that species differences in GnRH gene expression appear to exist since GnRH gene transcription appears to undergo much greater developmental changes in neonatal mice (46, 47, 48) compared with the observations in the present study on rats.

The developmental changes in GnRH gene expression observed in the present study are quite small, and thus it is unlikely that the regulation of GnRH mRNA levels in neonatal animals is primarily due to a glutamatergic influence. Indeed, changes in NR1 and GnRH RNA levels did not occur in parallel in either males or females. NR2b mRNA levels increased from P0 to P5 in parallel with GnRH mRNA in males but not in females, and decreased from P5 to P10 in parallel with GnRH mRNA in females but not males. However, we do not believe that these changes in GnRH mRNA are a direct reflection of alterations of NMDA receptors. Indeed, it is probable that the neonatal GnRH system is subject to a prominent inhibitory tone from GABAergic neurons (49) that must be removed before the establishment of an excitatory tone from glutamate and other neurons to enable the onset of puberty. The GnRH neurosecretory system is in fact regulated by numerous neurotransmitters, neurotrophic factors and steroid hormones (reviewed in Refs. 48, 50), and it is in all likelihood the combination of all of these events that is responsible for ultimate levels of GnRH biosynthesis and release.

The results of Exp III, in which we evaluated the ability of the perinatal GnRH neurosecretory system to respond to a stimulation or blockade of the NMDA receptor, also indicate a relative lack of responsiveness of the neonatal GnRH system to glutamate inputs. These data are consistent with a lack of expression of NR1-immunoreactivity in GnRH neurons of perinatal rats, suggesting that any NMDA receptor-mediated effects on GnRH neurons are, at best, indirect. Interestingly, a significant effect of treatment with the NMDA agonist, NMA, was observed in P5 and P10 male rats, which responded to NMA with an increase in GnRH mRNA levels. NR2a mRNA was detected earlier in neonatal male than female rats, and differences in the combinations of the NMDA receptor subunits between males and females may play a role in the differential responsiveness of neonates of different sexes and ages to NMA. NMDA has been reported to stimulate LH release in female rats as young as P15 (51), and in male rats at least as young as P10 [younger male rats were not examined in that study (13)], indicating that a gonadotropin response as well as a GnRH gene expression response to NMDA receptor activation may develop earlier in male than in female rats. The lack of a stimulatory effect of NMA in female rats, as well as differences in GnRH gene expression between males and females in Exp I, may also be due, at least in part, to lower exposure of females to steroid hormones neonatally (42, 43, 52). In adult animals, the ability of NMDA to stimulate the GnRH neurosecretory system is high in the presence of steroid hormones (53, 54), but low or absent in animals with low levels of sex steroids. A similar phenomenon may be responsible for this sex difference in young animals in the present study, during a developmental period when males have been exposed to high levels of steroid hormones, whereas females have not had a comparable exposure (42, 43, 52). In future studies, we will test this by examining GnRH gene expression, and its response to NMA, in neonatally androgenized female rats.

The effects of NMA on the stimulation of GnRH mRNA in immature male rats are attributed to a posttranscriptional mechanism such as altered mRNA stability because GnRH primary transcript levels were unaffected by NMA. We have previously reported a similar stimulatory effect of NMA on GnRH mRNA levels in adult male rats in the absence of a change in GnRH primary transcript levels (25). Other laboratories have reported that stimulation of the GnRH system with NMDA occurs in the absence of an increase in the immediate early gene c-fos expression in GnRH neurons (55, 56), supporting the idea that GnRH neurons do not become transcriptionally activated by NMDA receptor activation.

To determine whether NMDA receptor mRNA levels are reflected, at least qualitatively, by similar changes in protein, we evaluated the anatomical distribution of NR1 immunoreactivity, and its colocalization with GnRH neurons in developing rats in Exp IV. Quantitative analyses could not be performed due to large developmental differences in neuropil staining. Also, NR2a and 2b could not be examined in this manner due to a lack of suitable antibodies. For NR1, we observed that no GnRH neurons expressed this subunit in rats from P0 through P15. In a previous study, we reported that while few (about 2%) GnRH neurons coexpressed NR1 in P21 and P35 female rats, a significant increase in double-labeling occurred in P41 rats (19%), an age group that has already undergone puberty (16). Another laboratory reported that approximately 8% of GnRH neurons express NR1 immunoreactivity (40). Therefore, we think that before adulthood, the stimulatory effects of glutamate on the GnRH system are mediated by interneurons, or other NMDA or nonNMDA glutamate receptor subunits, several of which are developmentally regulated (21, 22, 23). This is again consistent with observations that GnRH gene transcription is not stimulated by NMDA receptor agonists (25, 55, 56).

While GnRH neurons did not express NR1 immunureactivity, we observed qualitative changes in the distribution and density of the NR1 immunocytochemistry in the OVLT/POA, with a similar pattern in male and female rats. In P0 animals, NR1 immunostaining was sparse; it then increased at P5, maintaining similar levels at P10. There was a decline in immunoreactivity from P10 to P15. NR1 immunoreactivity was also localized in patches beginning at P5, and most immunoreactivity was in the ventricular and ventral regions of the OVLT/POA. This increase in NR1 immunoreactivity through P10 occurred essentially in parallel with the changes in mRNA levels reported in Exp I. However, the decrease in protein immunoreactivity from P10 to P15 is not consistent with the maintained levels of NR1 mRNA, suggesting an uncoupling of mRNA and protein levels, or transport of the protein to neuronal processes.

The present study supports the idea that there is greater potential for the POA-AH to respond to changes in glutamate due to increases in the biosynthesis of NMDA receptors. The observations that the most profound changes in NR1 mRNA occur during the early postnatal period, before the onset of puberty, that NR2a mRNA levels differ between male and female rats, and that NR2b mRNA increase developmentally from P0 to P5 support our hypothesis that the establishment of the proper stimulatory environment that is necessary for the onset of puberty occurs during this early developmental stage. The mature NMDA receptors can then mediate the effects of increases in endogenous glutamate release that have been reported to occur subsequently, during puberty (53), and that probably play a role in the timing of the onset of puberty.


   Acknowledgments
 
The authors would like to thank Dr. John Morrison for the monoclonal antibody to NMDA-R1, and for his constant interest and support, Dr. Henryk Urbanski for the generous gift of the polyclonal antibody to GnRH, Dr. James Roberts for a critical reading of the manuscript, Dr. Deanna Benson for helpful discussions, Andrew P. Leonard for graphics, and Kim M. Longo for excellent technical assistance.


   Footnotes
 
1 All animal experiments were conducted in accord with Guidelines for the Care and Use of Experimental Animals, using protocols approved by the Institutional Animal Care and Use Committee at Mount Sinai School of Medicine (Grant No. 95–285NB). This work was supported by a Revson Foundation Fellowship and National Science Foundation Grant IBN-9723398 (to A.C.G.). A preliminary version of this work was presented at the Society for Neuroscience Meeting, New Orleans, Louisiana, 1997 (Abstract 798.1). Back

Received July 22, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wray S, Hoffman G 1986 A developmental study of the quantitative distribution of LHRH neurons within the central nervous system of postnatal male and female rats. J Comp Neurol 252:522–531[CrossRef][Medline]
  2. Silverman A-J, Antunes JL, Abrams GM, Nilaver G, Thau R, Robinson JA, Ferin M, Krey LC 1982 The luteinizing hormone-releasing hormone pathways in rhesus (Macaca mulatta) and pigtailed (Macaca nemestrina) monkeys: new observations on thick, unembedded sections. J Comp Neurol 211:309–317[CrossRef][Medline]
  3. Goldsmith PC, Song T 1987 The gonadotropin-releasing hormone containing ventral hypothalamic tract in the fetal rhesus monkey (Macaca mulatta). J Comp Neurol 237:130–149
  4. Watanabe G, Terasawa E 1989 In vivo release of luteinizing hormone releasing hormone increases with puberty in the female rhesus monkey. Endocrinology 125:92–99[Abstract]
  5. Urbanski HF, Ojeda SR 1990 A role of N-methyl-D-aspartate (NMDA) receptors in the control of LH secretion and initiation of female puberty. Endocrinology 126:1774–1776[Abstract]
  6. Gay VL, Plant TM 1987 N-methyl-D,L-aspartate (NMA) elicits hypothalamic GnRH release in prepubertal male rhesus monkeys (Macaca mulatta). Endocrinology 120:2289–2296[Abstract]
  7. Bourguignon J, Gerard A, Mathieu J, Mathieu A, Franchimont P 1990 Maturation of the hypothalamic control of pulsatile gonadotropin-releasing hormone secretion at onset of puberty. I. Increased activation of N-methyl-D-aspartate receptors. Endocrinology 127:873–881[Abstract]
  8. Claypool LE, Watanabe G, Terasawa E 1990 Effects of electrical stimulation of the medial basal hypothalamus on the in vivo release of luteinizing hormone-releasing hormone in the prepubertal and peripubertal female monkey. Endocrinology 127:3014–3022[Abstract]
  9. Gore AC, Terasawa E 1991 A role for norepinephrine in the control of puberty in the female rhesus monkey, Macaca mulatta. Endocrinology 129:3009–3017[Abstract]
  10. Van den Pol AN, Wuarin J-P, Dudek FE 1990 Glutamate, the dominant excitatory transmitter in neuroendocrine regulation. Science 250:1276–1278[Abstract/Free Full Text]
  11. Brann DW 1995 Glutamate: a major excitatory transmitter in neuroendocrine regulation. Neuroendocrinology 61:213–225[Medline]
  12. Urbanski HF, Ojeda SR 1987 Activation of luteinizing hormone-releasing hormone release advances the onset of female puberty. Neuroendocrinology 46:273–276[Medline]
  13. Cicero TJ, Meyer ER, Bell RD 1988 Characterization and possible opioid modulation of N-methyl-D-aspartic acid induced increases in serum luteinizing hormone levels in the developing male rat. Life Sci 42:1725–1732[CrossRef][Medline]
  14. Ondo JG, Wheeler DD, Dom RM 1988 Hypothalamic site of action for N-methyl-D-aspartate (NMDA) on LH secretion. Life Sci 43:2283–2286[CrossRef][Medline]
  15. Petersen SL, McCrone S, Keller M, Gardner E 1991 Rapid increases in LHRH mRNA levels following NMDA. Endocrinology 129:1679–1681[Abstract]
  16. Gore AC, Wu T, Rosenberg JJ, Roberts JL 1996 Gonadotropin-releasing hormone and NMDA-R1 gene expression and colocalization change during puberty in female rats. J Neurosci 16:5281–5289[Abstract/Free Full Text]
  17. Smyth C, Wilkinson M 1994 A critical period for glutamate receptor-mediated induction of precocious puberty in female rats. J Neuroendocrinol 6:275–284[CrossRef][Medline]
  18. Reier PJ, Cullen MJ, Froelich JS, Rothchild I 1977 The ultrastructure of the developing medial preoptic nucleus in the postnatal rat. Brain Res 122:415–436[CrossRef][Medline]
  19. Marani E, Rietveld WJ, Boon ME 1982 Monosodium glutamate accelerates migration of hypothalamic perikarya at puberty. Histochemistry 75:145–150[CrossRef][Medline]
  20. Constantine-Paton M 1990 NMDA receptor as a mediator of activity-dependent synaptogenesis in the developing brain. Cold Spring Harb Symp Quant Biol 50:431–443
  21. Franklin SO, Elliott K, Zhu Y-S, Wahlestedt C, Inturrisi CE 1993 Quantitation of NMDA receptor (NMDAR1) mRNA levels in the adult and developing rat CNS. Mol Brain Res 19:93–100[Medline]
  22. Sheng M, Cummings J, Roldan LA, Jan YN, Jan LY 1994 Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. Nature 368:144–147[CrossRef][Medline]
  23. Zhong J, Carrozza DP, Williams K, Pritchett DB, Molinoff PB 1995 Expression of mRNAs encoding subunits of the NMDA receptor in developing rat brain. J Neurochem 64:531–539[Medline]
  24. Jakubowski M, Roberts JL 1992 Multiplex solution hybridization-ribonuclease protection assay for quantitation of different ribonucleic acid transcripts from snap-frozen neuroendocrine tissues of individual animals. J Neuroendocrinol 4:79–89
  25. Gore AC, Roberts JL 1994 Regulation of gonadotropin-releasing hormone gene expression by the excitatory amino acids kainic acid and N-methyl-D,L-aspartate in the male rat. Endocrinology 134:2026–2031[Abstract]
  26. Jakubowski M, Blum M, Roberts JL 1991 Postnatal development of gonadotropin-releasing hormone and cyclophilin gene expression in the female and male rat brain. Endocrinology 128:2702–2708[Abstract]
  27. Yeo TTS, Gore AC, Jakubowski M, Dong K, Blum M, Roberts JL 1996 Characterization of gonadotropin-releasing hormone gene transcripts in a mouse hypothalamic neuronal GT1 cell line. Mol Brain Res 42:255–262[Medline]
  28. Monyer H, Sprengel R, Schoepfer R, Herb A, Higuchi M, Lomeli H, Burnashev N, Sakmann B, Seeburg PH 1992 Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 256:1217–1221[Abstract/Free Full Text]
  29. Sucher NJ, Akbarian S, Chi CL, Leclerc CL, Awobuluyi M, Deitcher DL, Wu MK, Yuan JP, Jones EG, Lipton SA 1995 Developmental and regional expression pattern of a novel NMDA receptor-like subunit (NMDAR-L) in the rodent brain. J Neurosci 15:6509–6520[Abstract/Free Full Text]
  30. Urbanski HF, Kim SO, Connolly ML 1990 Influence of photoperiod and 6-methoxybenzoxazolinone on the reproductive axis of inbred LSH/Ss Lak male hamsters. J Reprod Fertil 90:157–163[Abstract/Free Full Text]
  31. Siegel SJ, Brose N, Janssen WG, Gasic GP, Jahn R, Heinemann SF, Morrison JH 1994 Regional, cellular and ultrastructural distribution of the glutamate receptor subunit NMDAR1 in monkey hippocampus. Proc Natl Acad Sci USA 91:564–568[Abstract/Free Full Text]
  32. Huntley GW, Vickers JC, Janssen W, Brose N, Heinemann SF, Morrison JH 1994 Distribution and synaptic localization of immunocytochemically identified NMDA receptor subunit proteins in sensory-motor and visual cortices of monkey and human. J Neurosci 14:3603–3619[Abstract]
  33. Gore AC, Roberts JL 1995 Regulation of gonadotropin-releasing hormone gene expression in the rat during the luteinizing hormone surge. Endocrinology 136:889–896[Abstract]
  34. Nakanishi S 1992 Molecular diversity of glutamate receptors and implications for brain function. Science 258:597–603[Abstract/Free Full Text]
  35. Nyberg CL, Srivastava V, Hiney J, Lara F, Dees WL 1995 N-methyl-D-aspartic acid receptor mRNA levels and luteinizing hormone release in immature female rats: effects of stage of pubertal development and exposure to ethanol. Endocrinology 136:2874–2880[Abstract]
  36. Zuo Z, Mahesh VB, Zamorano PL, Brann DW 1996 Decreased gonadotropin-releasing hormone neurosecretory response to glutamate agonists in middle-aged female rats on proestrus afternoon: a possible role in reproductive aging? Endocrinology 137:2334–2338[Abstract]
  37. Riva MA, Tasceda F, Molteni R, Racagni G 1994 Regulation of NMDA receptor subunit mRNA expression in the rat brain during postnatal development. Mol Brain Res 25:209–216[Medline]
  38. Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH 1994 Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12:529–540[CrossRef][Medline]
  39. Portera-Caillau C, Price DL, Martin LJ 1996 N-methyl-D-aspartate receptor proteins NR2A and NR2B are differentially distributed in the developing rat central nervous system as revealed by subunit-specific antibodies. J Neurochem 66:692–700[Medline]
  40. Eyigor O, Jennes L 1996 Identification of glutamate receptor subtype mRNAs in gonadotropin-releasing hormone neurons in rat brain. Endocrine 4:133–139
  41. Eyigor O, Jennes L 1997 Expression of glutamate receptor subunit mRNAs in gonadotropin-releasing hormone neurons during the sexual maturation of the female rat. Neuroendocrinology 66:122–129[Medline]
  42. Barraclough CA 1961 Production of anovulatory sterile rats by single injections of testosterone proprionate. Endocrinology 68:62–67
  43. Donovan BT, ter Haar MB, Lockhart AN, MacKinnon PCB, Mattock JM, Peddie MJ 1975 Changes in the concentration of luteinizing hormone in plasma during development in the guinea pig. J Endocrinol 64:511–520[Abstract/Free Full Text]
  44. Chiappa SA, Fink G 1977 Releasing factor and hormonal changes in the hypothalamic-pituitary-gonadotrophin and adrenocorticotrophin systems before and after birth and puberty in male, female and androgenized female rats. J Endocrinol 72:211–224[Abstract/Free Full Text]
  45. Dohler KD, Wuttke W 1974 Serum LH, FSH, prolactin and progesterone from birth to puberty in female and male rats. Endocrinology 94:1003–1008[Medline]
  46. Wolfe AM, Wray S, Westphal H, Radovick S 1996 Cell-specific expression of the human gonadotropin-releasing hormone gene in transgenic animals. J Biol Chem 271:20018–20023[Abstract/Free Full Text]
  47. Gore AC, Roberts JL, Gibson MJ 1999 Mechanisms for the regulation of gonadotropin-releasing hormone gene expression in the developing mouse. Endocrinology 140:2280–2287[Abstract/Free Full Text]
  48. Gore AC, Roberts JL 1997 Regulation of GnRH gene expression in vivo and in vitro. Front Neuroendocrinol 18:209–245[CrossRef][Medline]
  49. Mitsushima D, Hei DL, Terasawa E 1994 {gamma}-aminobutyric acid is an inhibitory neurotransmitter restricting the release of luteinizing hormone-releasing hormone before the onset of puberty. Proc Natl Acad Sci USA 91:395–399[Abstract/Free Full Text]
  50. Sagrillo CA, Grattan DR, McCarthy MM, Selmanoff M 1996 Hormonal and neurotransmitter regulation of GnRH gene expression and related reproductive behaviors. Behav Genet 26:241–277[CrossRef][Medline]
  51. MacDonald MC, Wilkinson M 1992 Characterization and ontogenesis of N-methyl-D-aspartate-evoked luteinizing hormone secretion in immature female rats. J Neuroendocrinol 4:223–229
  52. Pang SF, Tang F 1984 Sex differences in the serum concentrations of testosterone in mice and hamsters during their critical periods of neural sexual differentiation. J Endocrinol 100:7–11[Abstract/Free Full Text]
  53. Goroll D, Arias P, Wuttke W 1993 Preoptic release of amino acid neurotransmitters evaluated in peripubertal and young adult female rats by push-pull perfusion. Neuroendocrinology 58:11–15[CrossRef][Medline]
  54. Carbone S, Szwarcfarb B, Losada M, Moguilevsky JA 1995 Effect of ovarian hormones on the hypothalamic excitatory amino acids system during sexual maturation in female rats. Neuroendocrinology 61:235–242[Medline]
  55. Lee W-S, Abbud R, Hoffman GE, Smith MS 1993 Effects of N-methyl-D-aspartate receptor activation on cFos expression in luteinizing hormone-releasing hormone neurons in female rats. Endocrinology 133:2248–2254[Abstract]
  56. Saitoh Y, Silverman A-J, Gibson MJ 1991 Norepinephrine neurons in mouse locus coeruleus express c-fos protein after N-methyl-D,L-aspartic acid (NMDA) treatment: relation to LH release. Brain Res 561:11–19[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Adams, M. M.
Right arrow Articles by Gore, A. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Adams, M. M.
Right arrow Articles by Gore, A. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals