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BOR - Papers in Press, published online ahead of print April 28, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.027946
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BIOLOGY OF REPRODUCTION 71, 676–683 (2004)
DOI: 10.1095/biolreprod.104.027946
© 2004 by the Society for the Study of Reproduction, Inc.

Neuroendocrinology

Regulation of N-Methyl-D-Aspartate Receptor Subunit Expression in the Fetal Guinea Pig Brain1

Dawn Owen3, Elaine Setiawan3, Antai Li3, Lucy McCabe3, and Stephen G. Matthews2,3,4,5

Departments of Physiology,3 Obstetrics and Gynecology,4 Medicine,5 Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
N-methyl-D-aspartate receptors (NMDARs) are critical for neuronal maturation and synaptic formation as well as for the onset of long-term potentiation, a process critical to learning and memory in postnatal life. In the current study, we demonstrated that NMDAR subunits undergo spatial, temporal, and sex-specific regulation. During development, we observed increasing NR1 and NR2A expression at the same time as levels of NR2B subunits decreased in the hippocampus and cortex in the fetal guinea pig. We have also shown that glucocorticoids can modulate fetal NMDAR subunit expression in a sex-specific fashion. This is clinically important because synthetic glucocorticoids are administered to pregnant women at risk of preterm labor. Repeated exposure to exogenous glucocorticoids caused a dose-dependent decrease in NR1 mRNA levels and increased NR2A mRNA expression in the female hippocampus at Gestational Day 62. There are significant changes in NMDAR subunit expression in late gestation. It is possible that these alter NMDA-dependent signaling at this time. Prenatal exposure to exogenous glucocorticoids modifies the trajectory of NMDAR subunit expression in females but not in males.

central nervous system, developmental biology, neuroendocrinology, neurotransmitters, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
N-methyl-D-aspartate receptors (NMDAR) are critical mediators of long-term potentiation (LTP), the hippocampal process that underlies learning and memory [1]. The ontogeny of NMDAR expression and the onset of their function are also important for global brain development, including the induction of synapse formation and neuronal maturation [2, 3]. Pathology of NMDAR function and subsequent glutamate toxicity are associated with several neurodegenerative diseases, such as epilepsy, stroke, Parkinson disease, Huntington disease, and Alzheimer disease [4].

NMDARs are excitatory ligand-gated ion channels comprised of four or five subunits: at least two NR1 subunits, which are central to glutamate binding and NMDAR function, and any of the NR2 (A, B, C, D) or NR3 (A, B, C) subunits [5, 6], which confer distinct pharmacological and electrophysiological properties. Each subunit has a distinct spatiotemporal expression profile in the developing brain, indicating that different subunits are turned on or off to modulate the degree of excitatory response [7, 8]. Events that disrupt NMDAR subunit development may have lasting effects on brain function in adult life.

Developmental expression of the NMDAR subunits is regulated by several inputs, including the hypothalamic-pituitary-adrenal axis. Glucocorticoids (GCs) modulate many developmental events in the brain and can cause long-term changes in hippocampal NMDAR subunit expression in adult life. In the early postnatal rat, maternal deprivation, a potent stressor, significantly reduced hippocampal NR2A and NR2B mRNA levels in adulthood [9]. Thus, abnormally elevated GC exposure in the perinatal period may significantly impact hippocampal function in adulthood through programming of NMDAR subunit expression.

However, little is known of the direct effect of GCs on NMDAR expression during fetal life. Synthetic GCs are routinely used to treat pregnant women at risk of preterm delivery [10]. Although a single course is recommended, recent surveys in Britain and Australia indicate that many obstetricians have been prescribing multiple doses of GCs to treat threatened preterm birth [11, 12]. Unfortunately, there is no evidence that multiple doses are more effective than a single dose regimen [10, 11]. Further, repeated exposure to antenatal GCs may have permanent effects on neurodevelopment, including long-term effects on learning and memory [13]. The guinea pig, like the sheep and human and unlike the rat, gives birth to neurologically mature young [14]; this allows detailed analysis of the developmental regulation of the NMDA receptor system during fetal life. In the current study, we hypothesized that 1) the hippocampal expression of NR1, NR2A, and NR2B mRNA undergoes distinct spatiotemporal regulation in the guinea pig; 2) there is a close association between mRNA and mature protein for NMDAR subunits during late gestation and early postnatal life; and 3) prenatal exposure to synthetic GCs differentially alters the expression of NMDAR subunits in the hippocampus. The dose (1 mg/kg) of synthetic GC used in this study in the guinea pig is comparable with the dose used in pregnant women (approximately 0.25 mg/kg) [10] because the guinea pig GR has a fourfold lower affinity for dexamethasone [15].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Development Female guinea pigs were mated in our animal facility as described previously [16]. This method produces accurately time-dated pregnant guinea pigs. These studies were performed using protocols approved by the Animal Care Committee at the University of Toronto and in accordance with the Canadian Council for Animal Care. Pregnant guinea pigs were killed by decapitation and fetuses collected on Gestational Days (GD) 40, 50, and 64 (term = 68 days). One group of guinea pigs was allowed to deliver naturally and neonates were killed by decapitation on Postnatal Day (PND) 7. Brains were removed and hemisected, with the left hemisphere frozen for in situ hybridization. Hippocampi were dissected from the right hemispheres for Western blot analysis. All tissues were stored at –80°C until processing. Normal litter size is 2–3 fetuses, and where possible, one male and one female fetus were taken from each litter for subsequent analysis.

Effect of Glucocorticoid in the Late Gestation Fetus

Pregnant guinea pigs were subcutaneously injected with betamethasone (Beta1; 1 mg/kg), dexamethasone (Dex1; 1 mg/kg), dexamethasone (Dex10; 10 mg/kg), or vehicle (saline [70%]/propylene glycol [30%], 200 µl; control) on GD 40/41 (period of neurogenesis), GD 50/51 (period of peak brain growth), and GD 60/61 (period of myelination) of gestation [14]. Animals were killed on GD 62 by decapitation and fetuses were collected. Brains were removed and frozen for in situ hybridization.

In Situ Hybridization

The method for in situ hybridization has been described in detail elsewhere [16]. Coronal cryosections (10 µm) were mounted onto poly-L-lysine coated slides, dried, and fixed in paraformaldehyde (4%). Oligonucleotide probes for NR1, NR2A, and NR2B were labeled using terminal deoxynucleotidyl transferase (Gibco, Burlington, ON, Canada) and [35S]-dATP (1300 Ci/mmol; Perkin Elmer, Woodbridge, ON, Canada) to a specific activity of 1.0 x 109 cpm/µg. Labeled probe in hybridization buffer (200 µl) was applied to slides at a concentration of 1.0 x 103 cpm/µl. The antisense probes were complementary to bases 409–453 of the coding sequence of rat NR1 (GenBank sequence S39221), bases 4328–4372 of the coding sequence of human NR2A (GenBank sequence NM_000833; 100% homologous to rat and mouse NR2A), and bases 4109–4153 of the coding sequence of mouse NR2B (GenBank sequence NM_008171; 100% homologous to human and rat NR2B). The NR1 subunit has eight splice variants, but our probe was designed to detect total NR1 mRNA [17]. Slides were incubated overnight in a moist chamber at 42.5°C. After washing in 1x SSC (20 min at 23°C, then 35 min at 55°C), the slides were rinsed and dehydrated in ethanol. The slides were dried and exposed to autoradiographic film (Biomax MR; Kodak, Perkin Elmer). Films were developed using an automatic processor (exposure: hippocampal and cortical NR1, NR2B, NR2A; 7 days).

For in situ hybridization, brain sections were processed simultaneously to allow direct comparison between groups. The sections were exposed together with 14C-standards (American Radiochemical Company, St. Louis, MO) to ensure analysis in the linear range of the autoradiographic film. The relative optical density (ROD) of the signal on autoradiographic film was quantified, after subtraction of background values, using a computerized image analysis system (Imaging Research Inc., St. Catharines, ON, Canada) [16]. Briefly, the area of interest to be analyzed was manually outlined by an operator blinded to treatment. An average ROD was derived for each such area. The procedures have been described in detail elsewhere [18]. Levels of NR1, NR2A, and NR2B mRNA were measured in the hippocampus (CA1/2, CA3, CA4), dentate gyrus, and cerebral cortex. Mean RODs were determined by sampling at least four animals per group, six to nine sections per animal. Incubation of slides with sense probes showed no hybridization signal (data not shown).

Western Blot Analysis

Hippocampi were homogenized in ice-cold RIPA lysis buffer (100– 500 µl; 1% Triton X-100, 10% SDS v/v, 0.15 M NaCl, 15.4 mM Tris-HCl, 0.5% deoxycholic acid w/v, 1 µM Na orthovanadate, Roche mini-EDTA-free Protease Inhibitor Cocktail; pH = 8.0). The homogenate was centrifuged (4°C, 10 000x g, 10 min) and the resulting supernatant was recentrifuged. Protein concentration of the resultant supernatant was determined by the Bradford method [19]. 2x Laemmli sample buffer (15 µl; Sigma, Oakville, ON, Canada) was added to each sample (50 µg protein), which was then denatured (boiled 5 min at 95°C). Samples were separated by SDS-PAGE (8% resolving polyacrylamide gel) and transferred electrophoretically to a nitrocellulose membrane (Bio-Rad, Mississauga, ON, Canada).

Nitrocellulose membranes were blocked overnight (4°C) in skim milk (5% w/v) phosphate-buffered saline with Tween 20 (PBS-T). Membranes were washed with PBS-T and incubated with NR1 antibody (1:500; AB1516; Chemicon International, Temecula, CA) in 5% skim milk PBS-T (1 h, 23°C). Membranes were then washed in PBS-T and incubated with HRP-conjugated goat anti-rabbit IgG (1:5000; 1 h, 23°C; Perkin Elmer). Blots were washed in PBS-T and exposed to Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer) and bands were visualized by exposure to Kodak Blue X-OMAT film for 30 sec to 1 min (Perkin Elmer). Films were developed by an automatic processor. Membranes were stripped in Restore Western Blot Stripping buffer (20 ml, 30 min, 23°C; Pierce, MJS Bioynx, Mississauga, ON, Canada). The blots were blocked overnight in 5% skim milk PBS-T and incubated with an antibody for NR2A/B (1:500; AB1548; Chemicon International) as described above. The absolute optical density of NR1 and NR2A/B were analyzed with computerized imaging software. All NMDAR signals were standardized to the signal for tubulin (1:5000; antitubulin; Sigma). All Western blots were performed a minimum of four times for each animal. Data were pooled to derive a mean value for each animal. Expression levels are presented as a ratio of NR1 or NR2A/B to tubulin signal.

Statistical Analysis

Group data are presented as means ± SEM and were statistically analyzed using analysis of variance (ANOVA) followed by the Duncan method of post hoc comparison (Statistica; Statsoft Inc., Tulsa, OK). Statistical significance was set at P < 0.05. Significant interactions in two-way ANOVAs are indicated in the Results section. For the ontogeny study, two-way ANOVAs were conducted for the effects of sex and age, whereas for the glucocorticoid treatment study, two-way ANOVAs were used to determine the effects of sex and glucocorticoid treatment.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hippocampal NR1 Expression: Development

NR1 mRNA was present in the CA1–4 regions of the hippocampus and dentate gyrus, with highest levels in the hippocampal CA3 region (Fig. 1a). There was a significant overall age effect for NR1 mRNA in all of the hippocampal regions analyzed (P < 0.01) as determined by two-way ANOVA. In all hippocampal regions, post hoc analysis revealed that NR1 mRNA increased significantly at GD 64, compared with GD 40 (P < 0.05), remaining high in early postnatal life (Fig. 1b and Table 1). There were no sex differences in the expression of NR1 mRNA in any of the hippocampal subfields.



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  		FIG. 1. a) Representative illustration of NR1 mRNA levels in the brain of a GD 50 and GD 64 fetus following in situ hybridization. Incubation with a sense probe produced no signal. Bar in a = 2.5 mm (representative scale for pictures); b) NR1 mRNA levels in hippocampal region CA1/2 in female (solid bars) and male (open bars) fetuses and neonates. Data are expressed as mean relative optical density (ROD) ± SEM; c) Hippocampal NR1 protein in the fetus and neonate. Inset: a representative Western blot for NR1 protein (108 kDa) and tubulin (55 kDa). Data are expressed as mean NR1:tubulin ratio ± SEM. Broken line indicates term (~GD 68). Numbers in each bar denote the number of animals in each group. Same letters indicate significant differences (P < 0.05)


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  		TABLE 1. Hippocampal and cortical NR1 mRNA expression.*

Western analysis revealed one specific NR1 band at 108 kDa, which is consistent with previous reports [20, 21] (Fig. 1c, inset). The pattern of expression for the 108-kDa band correlated closely with NR1 mRNA levels in the CA1/2 region of the hippocampus (Fig. 1b), which represents the largest region in the hippocampus. NR1 protein expression was shown to increase significantly at GD 50 from GD 40 in males only (P < 0.01) by post hoc analysis (Fig. 1c). By GD 64, hippocampal NR1 levels had increased significantly from GD 40 (P < 0.05) after post hoc analysis in both sexes. There were no sex differences in NR1 protein expression at any point in gestation.

Hippocampal NR2A and NR2B Expression: Development

Expression of NR2A and NR2B mRNA were highest in hippocampal region CA1/2 for both sexes (Figs. 2a and 3a). Levels of NR2A mRNA increased dramatically in all hippocampal subfields between GD 40 and GD 64 in both sexes after post hoc analysis (P < 0.05), with levels remaining high through to the end of gestation and into postnatal life (Fig. 2b and Table 2). There was a significant overall age effect on NR2A mRNA expression in all hippocampal subfields (P < 0.01) after two-way ANOVA.



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  		FIG. 2. a) Representative illustration of NR2A mRNA levels in the brain of a GD 50 and GD 64 fetus following in situ hybridization. Bar in a = 3 mm (representative scale for pictures); b) NR2A mRNA levels in hippocampal region CA1/2 in female (solid bars) and male (open bars) fetuses and neonates. Data are expressed as mean relative optical density (ROD) ± SEM; c) Hippocampal NR2A protein in the fetus and neonate. Inset: a representative Western blot for NR2A/B protein (180 kDa) and tubulin (55 kDa). Data are expressed as mean NR2A/B:tubulin ratio ± SEM. Numbers in each bar denote the number of animals in each group. Same letters indicate significant differences (P < 0.05)



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  		FIG. 3. a) NR2B mRNA levels in hippocampal region CA1/2 in female (solid bars) and male (open bars) fetuses and neonates; b) NR2B cortical mRNA levels in the fetus and neonate. Data are expressed as mean relative optical density (ROD) ± SEM. Broken line indicates term (~GD 68). Numbers in each bar denote the number of animals in each group. Same letters indicate significant differences (P < 0.05)


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  		TABLE 2. Hippocampal and cortical NR2A mRNA expression.*

In contrast, NR2B mRNA levels were unchanged throughout gestation and in early postnatal life in the CA1/ 2 and CA3 regions and dentate gyrus (Fig. 3a and Table 3). There was an overall age effect on CA4 mRNA expression (P < 0.01). In this region, post hoc analysis confirmed that NR2B mRNA levels decreased significantly at GD 64 compared with GD 40, but only in males (P < 0.05). By PND 7, both sexes showed reduced expression of NR2B mRNA in the CA4 subfield (P < 0.05). There were no individual sex differences in NR2B mRNA expression in any of the hippocampal regions examined.


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  		TABLE 3. Hippocampal NR2B mRNA expression.*

Western analysis revealed a single comigrating band for NR2A and NR2B that approximated the expected 170–180-kDa molecular masses of NR2A and NR2B [22] (Fig. 2c). The pattern of expression for the NR2A/B band corresponded with NR2A mRNA expression in the CA1/2 regions of the hippocampus but did not reflect NR2B mRNA levels. There was a significant age effect on NR2A/B protein expression (P < 0.01). NR2A/B protein levels were greatly elevated between GD 40 and GD 50, and the effect was greatest in males (P < 0.01) after post hoc analysis (Fig. 2c). However, by GD 64, both male and female fetuses showed increased expression of NR2A/B (P < 0.01). There were no sex differences in NR2A/B protein expression at any point in gestation.

Cortical NMDAR mRNA Expression: Development

NMDAR subunit mRNA expression was analyzed in the lateral parietal cortex as it was in the same anatomical section as the hippocampus. Cortical NR1 and NR2A mRNA expression followed the same pattern as the hippocampus, with a significant increase at GD 64 from GD 40 in both sexes (P < 0.05) after post hoc analysis (Tables 1 and 2). There was an overall age effect on the expression of all of the NMDAR subunits examined (P < 0.01). Interestingly, cortical NR2B mRNA levels decreased by 50% from GD 40 to GD 50, remaining low throughout late gestation and in the postnatal period (P < 0.003) on post hoc analysis (Fig. 3b).

Effect of Glucocorticoid Exposure on Fetal NR1 and NR2A Expression

There was no effect of repeated betamethasone exposure (1 mg/kg) on NR1 expression in either sex at GD 62 (Fig. 4, a and b). There was a dose-dependent decrease in NR1 mRNA expression in all hippocampal subfields at GD 62 after repeated dexamethasone treatment, but this effect was confined to female fetuses (P < 0.05) (Fig. 4, b and c). Repeated exposure to high-dose (10 mg/kg) dexamethasone injection significantly reduced NR1 mRNA levels in all hippocampal regions in female fetuses, while the lower dose (1 mg/kg) decreased expression in region CA3 and the dentate gyrus.



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  		FIG. 4. NR1 mRNA levels in the hippocampus and cortex of (a) GD 62 male fetuses; b) GD 62 female fetuses. Vehicle (solid bars), Beta1 (betamethasone 1 mg/kg; open bars), Dex1 (dexamethasone 1 mg/kg; shaded bars), Dex10 (dexamethasone 10 mg/kg; hatched bars); c) Representative illustration of NR1 mRNA levels in GD 62 female fetuses treated with vehicle (VEH), Dex1, and Dex10. Bar in c = 5 mm (representative scale for pictures). Data are expressed as mean relative optical density (ROD) ± SEM. Broken line indicates term (~GD 68). VEH (males, N = 6; females, N = 5), Beta1 (males, N = 5; females, N = 5), Dex1 (males, N = 5; females, N = 6), Dex10 (males, N = 7; females, N = 7). *, indicates significance from the vehicle control (P < 0.05)

There was an overall treatment effect on NR2A mRNA expression in hippocampal region CA1/2 and the dentate gyrus (P < 0.05) on two-way ANOVA. In the dentate gyrus, betamethasone treatment significantly increased NR2A levels (P < 0.05), but only in females, although there was a strong trend toward increased expression in males (P = 0.08) after post hoc analysis (Table 4). Neither dose of dexamethasone significantly affected NR2A expression in the hippocampus. Cortical NR1 and NR2A mRNA levels were unaltered by glucocorticoid treatment (Fig. 4 and Table 4). NR2B mRNA levels were not measured at GD 62 following repeat-course GC treatment due to limited tissue availability.


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  		TABLE 4. Hippocampal and cortical NR2A mRNA expression in glucocorticoid-exposed GD 62 fetuses.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study, we have demonstrated for the first time that the development of the NMDAR subunits in the fetal brain undergoes spatial, temporal, and sex-specific regulation. NR1 and NR2A mRNA and protein levels increase significantly near term in the fetal brain, remaining highly expressed in early postnatal life. In contrast, NR2B mRNA levels decreased significantly in the fetus in hippocampal subfield CA4 and the cerebral cortex. To date, no study has examined the influence of circulating GCs on NMDAR subunit expression in the fetal brain. Our study is the first to quantify specific changes in various NMDAR subunits following different synthetic GC (sGC) regimens. Administration of sGC had robust sex-specific effects on NR1 and NR2A mRNA expression in the late gestation fetal brain. In the female fetus at GD 62, there was a dose-dependent effect of dexamethasone on NR1 mRNA levels in all hippocampal subfields. Conversely, NR2A mRNA levels in the dentate gyrus of female fetuses were upregulated by betamethasone. None of the GC regimens affected NMDAR subunit expression in male fetuses; cortical NMDAR levels in both sexes were unaffected by GCs. Thus, it appears that GC exposure has sex-specific and region-specific effects on NMDAR subunit expression in the fetal brain.

As the NR1 subunit is critical to NMDAR activity, changes in NR1 subunit expression reflects an altered number of functional NMDARs [22, 23]. Our results show that hippocampal NR1 expression increases near term and postnatally. This parallels changes in total NMDA binding sites in the fetal guinea pig brain [24, 25]. Importantly, we have shown that hippocampal NR1 protein follows NR1 mRNA expression in the late gestation guinea pig fetus. This pattern is analogous to reports of increased NR1 expression and NMDA receptor binding in the early postnatal rat [7, 8, 17, 26–28]. In sheep, a species where extensive neurodevelopment occurs in utero, like the guinea pig and human, NMDA receptor binding density peaks in late prenatal and early postnatal life [29]. Of note, none of these studies has investigated molecular regulation of the different NMDAR subunits, or the relationship between mRNA and protein during development. Limited human studies of NMDA binding and NMDAR subunit expression have shown transient increases in midgestation followed by a decline in the perinatal period, though no detailed time course information is available [30–34]. Elevated NR1 mRNA levels and NMDA binding are associated with maturation-dependent increases in ion conductance and evoked potentials in the rat [35]. The onset of electrical activity occurs at GD 50 in the fetal guinea pig [36, 37], which precedes our demonstrated increase in NR1 expression in the perinatal period (GD 64). Increased functional NMDARs may also be involved in activity-dependent development of synapses and neuronal migration [5, 29]. Thus, events that disrupt gestational NR1 expression may have profound consequences on brain maturation and learning and memory in adult life.

In the current study, we show that NR2A mRNA levels increase dramatically in the limbic system and cerebral cortex near term in the fetal guinea pig and remain very high in postnatal life. In contrast, levels of NR2B mRNA do not change in the dentate gyrus or regions CA1–3 of the hippocampus during late gestation but decrease in hippocampal subfield CA4 and in the cerebral cortex. Our Western blots for NR2A/B protein revealed a comigrating band for both subunits. Gestational changes in NR2A/B protein levels corresponded very closely to NR2A mRNA levels. This is not surprising because the CA1 subfield and the dentate gyrus represent major components of the hippocampus and clearly indicate that changes in NR2A mRNA are closely linked to protein expression. NR2A is absent during fetal life in the rat, and there is a sudden onset of expression at birth, with increased levels in early postnatal life [7]; this is consistent with the differences in brain maturation at birth between the rat and the guinea pig [14]. The switch in expression to increased NR2A relative to NR2B is thought to alter receptor kinetics at a critical time in neuronal development [5, 7]. Increased contribution of the NR2A subunit results in NMDARs with rapidly decaying NMDAR-excitatory postsynaptic currents (EPSCs) that may allow neuronal circuits to develop experience-dependent synaptic plasticity [5, 38, 39]. Indeed, NR2A knockout mice are viable but show impaired spatial and contextual learning [1, 40] The higher expression of NR2B subunits in early fetal life may be critical for initiating activity-dependent neuronal development through the formation of channels with slow-deactivating currents [5, 41]. A role for NR2B in this context is supported by studies in NR2B knockout mice, which die in the early neonatal period [42]. Pathological changes in the ontogenic expression of NR2A may have long-term implications for learning and memory in adult life, while altered NR2B expression may have significant impact on normal neuronal development.

The ontogeny of the NMDAR subunits is largely regulated by the level of synaptic activity and various trophic factors [2, 5]. However, circulating hormones, such as GCs, can modulate the expression of NMDARs and alter the course of brain development. These concerns are highly relevant to the clinical setting where pregnant women who, at risk of delivering a preterm fetus, are administered synthetic GCs to mature the fetal lungs [10]. Many of these women have received multiple doses of GCs, without clinical evidence to support such practice [10, 11]. Previous studies in primates and sheep have shown that prenatal exposure to exogenous GCs causes dose-dependent damage to developing neurons, resulting in neuronal size reduction, neuronal loss, and dendritic atrophy; these detrimental effects are thought to result from excitotoxicity mediated by glutamate and NMDARs [43–46]. However, there is virtually no information on the direct impact of prenatal GCs on NMDAR development. In the current study, we have shown that repeated prenatal administration of GCs has sex-specific effects on NMDAR subunit expression in late gestation. We report a dose-dependent reduction of NR1 mRNA levels in the fetal guinea pig hippocampus, an effect that only occurs in females. This significant decrease in NR1 subunits in the perinatal period may have implications on the number of functional NMDARs at a time when NMDAR ion flux is maturing. Further, if these effects are permanent, learning and memory processes may be affected in postnatal life. In this context, we have recently reported that repeated prenatal exposure to synthetic GCs, at the same time points as described in the present study, results in altered stress-related behavior in juvenile guinea pigs [47]. We also observed increased NR2A mRNA levels in the female dentate gyrus following repeated GC exposure. The significance of increased NR2A expression when NR2A levels are normally peaking in the fetal hippocampus remains to be determined. There is some specificity of the type of exogenous GC used, as NR1 expression was altered by dexamethasone while NR2A was only affected by betamethasone treatment. The reason for this glucocorticoid-specific difference is not known, but may be of clinical significance because dexamethasone is the drug of choice for prenatal therapy in Europe, while betamethasone is more widely used in North America [10]. The mechanism by which glucocorticoids can modulate NMDAR expression is unclear. It is possible that glucocorticoids can exert direct transcriptional effects on NMDAR subunit genes, but to our knowledge, none of the NMDAR genes have been investigated for a glucocorticoid-response element. Ligand-bound glucocorticoid receptors can also recruit other transcription factors, which in turn may alter NMDAR subunit composition. In addition, glucocorticoids can influence NMDA-dependent Ca2+ ion flux in cultured hippocampal neurons [48]. Disturbance of neuronal membrane potentials in the fetus may present a nongenomic route by which prenatal glucocorticoids can modify activity-dependent NMDAR subunit expression. Further, prenatal glucocorticoids may interact with gonadal endocrine axes to produce sex-specific outcomes on NMDAR subunit composition. In the guinea pig, development of sexual dimorphism in the brain occurs prenatally and depends on the relative concentration of circulating androgens in mid- to late gestation [49]. Further investigation is needed to elucidate the connection between glucocorticoids and NMDAR expression.

In conclusion, NMDAR subunit composition is subject to complex regulation during fetal development. The changes in NMDAR subunit expression correlate with key events in neuronal maturation and synapse formation. The number of functional NMDARs increases with gestational age as NR1 subunit expression rises near term. The relative contribution of NR2A to NMDAR heteromers appears to increase at the same time that NR2B decreases, giving rise to a fetal switch that likely facilitates experience-dependent synaptic plasticity to occur. Furthermore, the immature NMDAR system is modulated by exposure to GCs in fetal life. Repeated administration of prenatal GCs caused sex-specific, dose-dependent reductions in perinatal hippocampal NR1 expression, which may lead to decreased NMDAR function and impaired learning and memory in adult female offspring. Understanding the development of NMDAR subunit expression and their regulation will provide insight into many aspects of neuronal development as well as postnatal processes, such as learning and memory.


   ACKNOWLEDGMENTS
 
We would like to thank Ms. Sonja Banjanin and Ms. Alice Kostaki for their assistance in these experiments.


   FOOTNOTES
 
1 Supported by the Canadian Institute for Health Research (CIHR) Premier Research Excellence Award (PREA) to S.G.M. (MOP-49511), and a CIHR MD/PhD Studentship to D.O. Back

2 Correspondence: S.G. Matthews, Department of Physiology, Faculty of Medicine, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada. FAX: 416 978 4940; stephen.matthews{at}utoronto.ca Back

Received: 4 February 2004.

First decision: 1 March 2004.

Accepted: 5 April 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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