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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
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Abstract |
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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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 [
-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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. ANOVA demonstrated a significant
change in NR1 mRNA levels with development (P <
0.0001) that was similar in both male and female rats (Fig. 2). 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.
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). 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.
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). With respect to
age, NR2b mRNA levels were significant lower at E18 and P0 compared
with P5 through P15.
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. For both sexes, GnRH mRNA levels
were developmentally regulated (P < 0.0001; Fig. 6). In males, GnRH mRNA levels increased
significantly from P0 to P5 (P < 0.05; Fig. 6a), and
in females, they decreased significantly from P5 to P10
(P < 0.05; Fig. 6b). For GnRH primary transcript
levels in the nucleus, there was also a significant developmental
effect (P < 0.0001; Fig. 7). While in male rats GnRH primary
transcript levels did not undergo significant developmental changes
(Fig. 7a), in females, these levels decreased developmentally from P5
to P10 (P < 0.01; Fig. 7b) in parallel with GnRH mRNA
levels. GnRH primary transcript levels in females then increased
significantly from P10 to P15 (P < 0.005; Fig. 7b).
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). In male rats, NMA caused a
significant stimulation of GnRH mRNA levels at P5 and P10
(P < 0.05; Table 1). 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 1). For GnRH primary transcript levels, there was no
significant effect of NMA or MK-801 in either male or female rats
(Table 1).
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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. 8a). 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. 8b). 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. 8a), 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Acknowledgments |
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Footnotes |
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Received July 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric acid is an inhibitory neurotransmitter restricting the
release of luteinizing hormone-releasing hormone before the onset of
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