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Chronic Ethanol Exposure Alters MK-801 Binding Sites in the Cerebral Cortex of the Near-Term Fetal Guinea Pig
Alcohol, Vol. 17, No. 3, pp. 215–221, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0741-8329/99/$–see front matter
PII S0741-8329(98)00050-0
Chronic Ethanol Exposure Alters MK-801 Binding Sites in the Cerebral Cortex of the Near-Term Fetal Guinea Pig J. CHIU,* J. F. BRIEN,* P. WU,† J. H. EUBANKS,‡ L. ZHANG‡ AND J. N. REYNOLDS* *Department of Pharmacology and Toxicology, Queen’s University, Ontario, Canada †Department of Pharmacology, University of Toronto, Toronto, Canada ‡Playfair Neuroscience Unit, Toronto Hospital Western Division, Toronto, Canada Received 19 February 1998; Accepted 20 July 1998 CHIU, J., J. F. BRIEN, P. WU, J. H. EUBANKS, L. ZHANG AND J. N. REYNOLDS. Chronic ethanol exposure alters MK-801 binding sties in the cerebral cortex of the near-term fetal guinea pig. ALCOHOL 17(3) 215–221, 1999.—The mechanism of ethanol central nervous system (CNS) teratogenesis, resulting from chronic maternal ingestion of high-dose ethanol during pregnancy, is not clearly understood. One of the target sites for ethanol-induced damage in the developing brain is the cerebral cortex. It has been proposed that chronic prenatal ethanol exposure alters NMDA receptors in the developing cerebral cortex. To test this hypothesis, timed pregnant guinea pigs were administered one of the following oral treatments throughout gestation: 4 g ethanol/kg maternal body weight/day; isocaloric sucrose/pair-feeding; water; or no treatment (ad lib). Near-term fetuses were studied at gestational day (GD) 63 (term, about GD 68). This ethanol regimen produced a maternal blood ethanol concentration of 66 6 4 mM (304 6 19 mg/dl) at 1 h after the daily dose on GD 58. The chronic ethanol regimen decreased near-term fetal body weight (12–26% decrease), brain weight (23% decrease), and cerebral cortical weight (21% decrease), compared with the isocaloric sucrose/pair-feeding, and combined water/ad lib experimental groups. Saturation analysis of near-term fetal cerebral cortical membranes using a [3H]MK-801 radioligand binding assay demonstrated a decreased affinity and increased number of MK-801 binding sites for the chronic ethanol regimen compared with the control treatments. These data support the suggestion that upregulation of NMDA receptors in the cerebral cortex after chronic prenatal ethanol exposure could lead to NMDA receptor-mediated excitotoxicity in this brain region. © 1999 Elsevier Science Inc. All rights reserved. Ethanol
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ment, including neuronal migration of cortical neurons (29), cell-cycle kinetics (31), and spine distribution of pyramidal cells (40). Conceivably, prenatal ethanol exposure can have deleterious effects on neurogenesis and synaptogenesis, thereby impairing neuronal function. Currently, there is no effective treatment for the neuronal defects induced by prenatal ethanol exposure, primarily because the mechanism of ethanol-induced damage of the developing CNS is not well understood. Recently, it was proposed that perturbation of synaptic transmission mediated by the N-methyl-D-aspartate (NMDA) receptor plays a key role in the mechanism of ethanol-induced damage of the developing CNS (36). The NMDA receptor is a subfamily of excitatory amino acid receptors of the L-gluta-
IT is well established that ethanol can injure the developing fetus. Chronic excessive ethanol (alcohol) consumption during pregnancy can lead to a specific pattern of birth defects called the fetal alcohol syndrome (FAS) (21). The primary features of FAS are growth restriction, craniofacial abnormalities, and central nervous system (CNS) dysfunction (10). Of all these defects, CNS dysfunction appears to be the most debilitating one, and its manifestations include motor incoordination, developmental delay, neurological abnormalities, and intellectual impairment. One of the target sites of ethanol in the developing brain is the cerebral cortex, which plays a key role in cognitive function and regulation of behaviour. Prenatal ethanol exposure can interfere with neuronal develop-
Requests for reprints should be addressed to Dr. J. N. Reynolds, Department of Pharmacology and Toxicology, Queen’s University, Kingston, Ontario K7L 3N6, Canada. Tel: (613) 533-6946; Fax: (613) 533-6412; E-mail: [email protected]
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mate pathway (32). It is a ligand-gated ion channel that has high permeability to calcium (26) and requires glycine as a coagonist (20,24). The NMDA receptor has many important roles during brain development; its activation promotes neuronal survival and differentiation (4), synaptogenesis (11), and formation of neuronal circuitry (35). Optimal activation of the L-glutamate–NMDA receptor system is considered to be essential for normal brain development; underactivation or overactivation of this system during development may lead to brain dysmorphology and dysfunction (26,36). Any substance perturbing NMDA receptor function, therefore, could interfere with developmental processes in the brain. In the hippocampus, another target site of ethanol-induced brain damage, chronic maternal ethanol administration decreased the number of NMDA-sensitive [3H]glutamate binding sites, but not their affinity, in the near-term fetal guinea pig (1). Similarly, prenatal ethanol exposure produced a decrease in NMDA-sensitive [3H]glutamate binding in the hippocampus of the adult rat (37). It has been reported that chronic prenatal ethanol exposure decreases NMDA-stimulated Ca21 entry in dissociated neurons obtained from neonatal rat frontal cortex (25), accompanied by decreased expression of specific subunit subtypes of the NMDA receptor complex (18,38). The present study was focused on testing the hypothesis that chronic prenatal ethanol exposure alters NMDA receptors in the developing cerebral cortex. The objective was to investigate, in the near-term fetal cerebral cortex, the effect of chronic prenatal ethanol exposure on NMDA receptor number and affinity. The guinea pig was selected as the experimental animal because its brain growth spurt occurs prenatally during the third-trimester-equivalent of gestation, which is more similar to the human situation compared with other rodent species, including the rat (13). The ethanol regimen used in this study has been shown previously to produce damage to the developing CNS (1,3,6,23). METHOD
Materials (1)-[3H]MK-801 (23.9 Ci/mmol) was supplied by DuPont New England Nuclear (Boston, MA). (1)-MK-801 maleate was obtained from Tocris Cookson (St. Louis, MO). All other chemicals were at least reagent grade, and were purchased from Sigma Chemical Co. or Fischer Scientific. Tap water was used to prepare the chemical cocktail for gas–liquid chromatographic (GLC) analysis of maternal blood ethanol concentration, because it has been found to be the most reliable source of water containing no GLC-interfering volatile organic compounds. All other aqueous solutions and buffers were prepared using deionized water obtained from a Barnstead Nanopure® system. Experimental Animals and Chronic Treatment Regimens Nulliparous Dunkin–Hartley strain guinea pigs were purchased from Charles River Canada Inc. (St. Constant, Quebec, Canada). All animals were kept in a room at an ambient temperature of 238C, with a 12-h light/12-h dark cycle with lights on at 0700 h. Female guinea pigs (500–600 g body weight) were bred with male guinea pigs (body weight . 700 g) of the same strain using an established procedure (14). The last day of full vaginal membrane opening was taken as gestational day (GD) 0. On GD 1, the pregnant animals were separated and housed singly in stainless steel wire cages. The
pregnant guinea pigs were assigned randomly to one of four experimental groups, comprising no treatment with ad lib acces to pellet food (Purina Guinea Pig Chow 5025®) and water, or one of the following three chronic treatments from GD 2 to GD 62 via intubation into the oral cavity: 4 g ethanol/kg maternal body weight/day with ad lib access to pellet food and water; isocaloric sucrose and pair-feeding to a particular ethanol-treated pregnant animal with ad lib access to water; or water with ad lib access to pellet food and water. Pair-fed animals were matched with an ethanol-treated animal, and given the same amounts of lab chow consumed by the ethanol-treated animal on each day of drug administration. This protocol ensured that ethanol-treated and isocaloric sucrose pair-fed animals received the same amount of calories on any given day during the dosing regimen. The ethanol (30% v/v) and sucrose (42% w/v) solutions were prepared with tap water. The ethanol, isocaloric sucrose, and water treatment regimens were administered as two equally divided volumes, given 2 h apart between 1100 and 1500 h. The pregnant animals were weighed and their vaginal membrane status was monitored daily. All animals were cared for according to the principles and guidelines of the Canadian Council on Animal Care. The experimental protocol was approved by the Queen’s University Animal Care Committee. Maternal Blood Ethanol Concentration Determination On GD 58, a maternal blood sample was collected from an ear vein at 1 h after the second divided volume of the daily dose of ethanol for the determination of ethanol concentration using an established GLC method involving head-space gas analysis (39). The blood ethanol concentration was determined in duplicate. Tissue Collection On GD 63, individual pregnant guinea pigs received no treatment and were killed by decapitation. The litter was delivered by cesarean section, and each fetus was decapitated. The fetal brain was excised, and the cerebral cortex was dissected. The body, brain, and cerebral cortical weights, and gender of each fetus were determined. The cerebral cortex was frozen immediately in liquid nitrogen, and was stored at 2708C until analyzed. Crude Plasma Membrane Preparation for Radioligand Binding Assay Each frozen cerebral cortex was thawed and homogenized in 20 ml/g tissue of buffer A (300 mM sucrose, 10 mM Tris, 2 mM EDTA, pH 7.3). The plasma membrane was obtained by differential centrifugation. In brief, the homogenate was centrifuged at 1000 3 g for 20 min at 48C, after which the supernatant was collected and centrifuged at 16,000 3 g for 1 h at 48C. The resulting pellet was collected and resuspended in 3 ml of buffer A. This membrane preparation was stored at 2708C. On the day of experimentation, an aliquot of the membrane suspension (8–10 mg protein) was thawed, diluted in 0.5 ml of buffer B (20 mM HEPES, 1 mM EDTA, pH 7.0), and incubated at 378C for 30 min. Following incubation, the suspension was centrifuged at 48C for 10 min at 13,000 3 g. The pellet was washed four times by resuspending it in 1 ml of buffer B and centrifuging at 48C for 5 min at 13,000 3 g. The final pellet was resuspended in buffer B to obtain a final crude plasma membrane preparation containing 2–4 mg membrane protein/ml.
CHRONIC ETHANOL EXPOSURE ALTERS MK-801 BINDING [3H]MK-801 Binding Assay An incubation mixture (final volume 5 0.5 ml) containing 50 ml of the final crude plasma membrane preparation (100– 200 mg protein) was incubated with one of nine different concentrations of [3H]MK-801 ranging from 0.2 to 45 nM. The binding assay was performed in the presence of 100 mM L-glutamate and 100 mM glycine. Preliminary experiments performed on control fetal cerebral cortex using lower concentrations (10 mM of each) of glutamate and glycine resulted in binding to two sites; one with high affinity and one with low affinity for [3H]MK-801 (data not shown). Higher concentrations (100 mM) of these two coagonists eliminated the lowaffinity component. Comparison of one- and two-site fits to the data using the F-test (Prism, Graphpad Software, Inc., San Diego, CA) indicated that a two-site model did not fit the data significantly better than a one-site model (p . 0.05). The higher concentrations of glutamate and glycine were therefore used for all subsequent experiments, and experimental data were fit to a one-site binding equation. Nonspecific binding was determined by incubation with 10 mM nonradioactive MK-801. The binding assay was conducted in duplicate at 378C for 3 h. Bound [3H]MK-801 radioactivity was determined by rapid vacuum filtration onto Whatman GF/B glass fibre filters presoaked with ice-cold buffer B. The filters were rinsed immediately with two 5-ml aliquots of ice-cold buffer B, and then placed in a scintillation vial containing 5 ml of scintillation cocktail followed by incubation in the dark at room temperature for 15 h. The radioactivity retained on the filter was quantified by liquid scintillation spectrometry using a Beckman LS3800® scintillation counter. The concentration of protein in the membrane preparation was determined using a protein-dye binding assay (5). Data Analysis The number (Bmax) and affinity (Kd) of the [3H]MK-801 binding sites of individual fetuses were determined from their individual saturation isotherm data using nonlinear regression analysis (Prism, Graphpad Software Inc.). The data are presented as group means 6 SEM, and were analysed by randomized-design one-way ANOVA followed by the Student–Newman–Keuls test for multiple comparisons (Prism, Graphpad Software Inc.). RESULTS
Maternal Blood Ethanol Concentration The chronic oral regimen of 4 g ethanol/kg maternal body weight/day produced a maternal blood ethanol concentration of 66 6 4 mM (304 6 19 mg/dl; n 5 10) at 1 h after the administration of the second divided volume of the daily ethanol dose on GD 58. A previous study of ethanol disposition in the near-term maternal–fetal unit of the pregnant guinea pig has demonstrated that the maternal and fetal blood ethanol concentrations are of similar magnitude and appear to be maximal at about 1 h after oral administration of the last equally divided volume of 4 g ethanol/kg maternal body weight (9). The maternal blood ethanol concentration of the present study is comparable with the data of our other investigations in the guinea pig, in which this chronic ethanol regimen produced CNS injury (1,3,6,23). Pregnancy Outcome Data Daily handling and administration of water had no effect on any of the parameters measured in this study. For this rea-
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son, the water treatment and ad lib groups were combined for the purposes of data analysis. There was no maternal lethality for the ethanol, isocaloric sucrose/pair-feeding, water and ad lib groups; however, there was one incident of embryonic reabsorption in each of the ethanol and water experimental groups. The litter sizes of the pregnant guinea pigs at GD 63 in the ethanol, isocaloric sucrose/pair-feeding, and water/ad lib groups were 3 6 0.4 (n 5 8 litters); 3.4 6 0.3 (n 5 8 litters), 3.4 6 0.3 (n 5 12 litters), respectively, and there was no difference among the three experimental groups. The total numbers of male and female offspring for the ethanol, sucrose, and water/ad lib groups were, respectively, 14 and 10 (n 5 8 litters), 13 and 14 (n 5 8 litters), and 19 and 22 (n 5 12 litters). There was no difference in the percentage of females and percentage of males per litter among the ethanol, sucrose, and water/ad lib groups: ethanol group, 62 6 7% for male, 39 6 7% for female; sucrose group, 45 6 11% for male, 55 6 11% for female; water/ad lib group, 50 6 8% for male, 50 6 8% for female. Effects of Ethanol on Fetal Body, Brain, and Cerebral Cortical Weights Both chronic ethanol and isocaloric sucrose/pair-feeding treatments decreased fetal body weight (Fig. 1A). Ethanol treatment produced a 12% decrease (p , 0.01) compared with sucrose treatment, and a 26% decrease (p , 0.001) compared with the combined water treatment/ad lib group. Sucrose treatment decreased fetal body weight by 16% (p , 0.01) compared with the water treatment/ad lib group. Chronic ethanol treatment decreased brain weight by 23% (p , 0.001) (Fig. 1B) and cerebral cortical weight by 21% (p , 0.001) (Fig. 1C) compared with the other experimental groups. There was no difference in the brain and cerebral cortical weight data between the sucrose and water treatment/ad lib groups. [3H]MK-801 Binding in the Fetal Cerebral Cortex MK-801 binding site number and affinity in the near-term fetal cerebral cortex were determined using saturation isotherm analysis with [3H]MK-801 as the radioligand. Saturation binding data for the three treatment groups, and a representative Scatchard plot from each of the experimental groups, are shown in Figure 2. The Bmax of MK-801 binding sites in the cerebral cortex was increased by chronic prenatal ethanol exposure (Figs. 2A, 3A) compared to the isocaloric sucrose and water/ad lib experimental groups. The Kd of the MK-801 binding sites for the ethanol group was increased by 57% (p , 0.001) compared with the other experimental groups. DISCUSSION
The regimen of 4 g ethanol/kg maternal body weight/day administered throughout gestation produced body, whole brain, and cerebral cortical growth restriction in the nearterm fetal guinea pig. Isocaloric sucrose/pair-feeding treatment only produced body growth restriction and did not affect whole brain and cerebral cortical weight, manifesting the “brain sparing effect” associated with undernutrition (33,34). These data demonstrate that undernutrition is not the mechanism underlying ethanol-induced alterations in MK-801 binding or weight of the cerebral cortex of the guinea pig; ethanol per se can produce these effects in this brain region. In the present study, [3H]MK-801 was utilized to examine the NMDA receptors in the near-term fetal guinea pig cere-
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CHIU ET AL. bral cortex. MK-801 is a selective, noncompetitive, reversible antagonist of the NMDA receptor. Because its binding site is in the ion channel (22,26), the value of maximum specific binding of [3H]MK-801 under equilibrium conditions is generally considered to be equivalent to the number of NMDA receptors. Channel opening is essential for the high-affinity specific binding of [3H]MK-801; therefore, saturating concentrations of L-glutamate and glycine were used in our binding assay to ensure maximum NMDA receptor activation. In the near-term fetal cerebral cortex, chronic maternal ethanol administration decreased the affinity of [3H]MK-801 binding sites and increased their number. Given the specific NMDA ion channel binding properties of nanomolar concentrations of MK-801, the data indicate that prenatal ethanol exposure increases the number of NMDA receptors in the near-term fetal guinea pig cerebral cortex. In contrast, chronic maternal ethanol administration decreased the number, but not the affinity, of NMDA-sensitive [3H]glutamate binding sites in the hippocampus of the near-term fetal guinea pig (1). Collectively, the data from the two studies demonstrate that chronic prenatal ethanol exposure alters NMDA receptors in both the cerebral cortex and hippocampus, but apparently by different mechanisms. Decreased number of NMDA receptors in the hippocampus after chronic prenatal ethanol exposure has been a consistent finding from several different laboratories (1,37,44). This effect of prenatal ethanol exposure on hippocampal NMDA receptors persists into the postnatal period (37), and may be a consequence of neuronal cell loss in the CA1 region of the hippocampus (30). The effects of chronic prenatal ethanol exposure on cerebral cortical NMDA receptors are not clearly understood. As the present manuscript was being prepared, another study was published (12) demonstrating that prenatal and early postnatal ethanol exposure in the rat decreased the number of [3H]MK-801 binding in a combined cerebral cortical/hippocampal preparation. This same group has also published data suggesting that chronic prenatal ethanol exposure decreases the expression of NR 2B subunit protein in the rat cerebral cortex, but has no effect on NR 1 subunit expression (18,38). The reason for the apparent discrepancy between the [3H]MK-801 binding data of our study (decreased affinity, increased number) and the recently published investigation of Diaz-Granados et al. (decreased number) (12), is uncertain. However, there are some significant methodological differences that make direct comparisons between the two studies very difficult. First, the developmental stage at which the animals were examined differed between the two studies. Whereas Diaz-Granados et al. (12) examined [3H]MK-801 binding in a combined hippocampal/cerebral cortical membrane preparation obtained from 20–23-day-old rats, we
FIG. 1. Body and brain weight data for near-term fetal guinea pigs. (A) Body weight of near-term fetuses of pregnant guinea pigs that received 4 g ethanol/kg maternal body weight/day (n 5 24), isocaloric sucrose/pair-feeding (n 5 27), or water/no treatment (ad lib) (n 5 41) from GD 2 to GD 62. **Indicates statistical difference (p , 0.05)
between ethanol and each of the other two experimental groups. *Indicates statistical difference (p , 0.01) between sucrose and the water/ad lib (AL) group. (B) Whole brain weight of near-term fetuses of pregnant guinea pigs of the three experimental groups described in (A) (ethanol, n 5 24; sucrose, n 5 27; water/ad lib, n 5 41). *Indicates statistical difference (p , 0.001) between ethanol and each of the other two experimental groups. (C) Cerebral cortical weights of near-term fetuses of pregnant guinea pigs of the three experimental groups described in (A) (ethanol, n 5 9; sucrose, n 5 11, water/ad lib, n 5 13). *Indicates statistical difference (p , 0.001) between ethanol and each of the other two experimental groups.
CHRONIC ETHANOL EXPOSURE ALTERS MK-801 BINDING
FIG. 2. [3H]MK-801 binding to cerebral cortical membranes prepared from near-term fetal guinea pigs. Animals were randomly selected from three different litters. (A) Saturation isotherms for [3H]MK-801 (0.2–45 nM) binding to cerebral cortical membranes in the presence of saturating concentrations of L-glutamate (100 mM) and glycine (100 mM). The best fit to the experimental data was used to determine the Kd and Bmax for [3H]MK-801 binding for ethanol (solid line), isocaloric sucrose pair-fed (hatched line), and water/ad lib groups (dashed line). (B) Representative Scatchard plots of [3H]MK-801 binding for each of the three experimental groups. A clear change in the slope of the ethanol-treated animal (solid line) and difference in the x-intercept reflects the increased Kd and Bmax in this group compared to both isocaloric sucrose pair-fed (hatched line) and water/ad lib animals (dashed line).
tested a cerebral cortical membrane preparation from the near-term fetal guinea pig. The ontogenic profile of NMDA receptor expression in the hippocampus and cerebral cortex differ dramatically between the rat and the guinea pig. In the rat, NMDA receptor expression in the hippocampus and cerebral cortex undergoes a transient upregulation that begins
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FIG. 3. Characteristics of [3H]MK-801 binding sites in the cerebral cortex of the near-term fetal guinea pig. (A) Bmax of the MK-801 binding sites in the cerebral cortex of near-term fetuses of the three experimental groups described in Fig. 1A (ethanol, n 5 9; isocaloric sucrose/pair-feeding, n 5 5; water/ad lib, n 5 6). The data are the mean 6 SEM of the Bmax values obtained from the saturation isotherms of the individual fetuses. Ethanol increased the Bmax values for [3H]MK-801 binding to near-term fetal guinea pig cerebral cortex. *Indicates statistical difference (p , 0.05) between ethanol and each of the other two experimental groups. (B) Kd of the specific binding of [3H]MK-801 to NMDA receptors in the cerebral cortex of nearterm fetuses of the three experimental groups. The data are the mean 6 SEM of the Kd values obtained from the saturation isotherms of the individual fetuses. *Indicates statistical difference (p , 0.001) between ethanol and each of the other two experimental groups.
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during the first postnatal week and peaks between postnatal days 9 and 15 (27,28,42). Thereafter, NMDA receptor density in these brain regions declines rapidly, reaching adult levels by postnatal days 20–28. In contrast, the transient increase in the density of NMDA receptors in the guinea pig hippocampus beings prenatally between GD 50 and GD 62 (2). NMDA receptor density in the guinea pig hippocampus peaks in the near-term fetus (around GD 62) and remains elevated until at least the second postnatal week, and thereafter declines towards adult levels. Second, in the present study we obtained cerebral cortical tissue from near-term fetal guinea pigs 24 h after the last dose of ethanol. Diaz-Granados et al. (12) used a combined hippocampal/cerebral cortical membrane preparation obtained from 20–23-day-old rats 11–14 days after the last dose of ethanol. This suggests the possibility that ethanol withdrawal may be an important determinant of ethanol-induced damage to the developing brain. Third, the assay conditions in which [3H]MK-801 binding was examined were different between the two studies. Diaz-Granados et al. (12) utilized a combined hippocampal/cerebral cortical membrane preparation, and carried out the binding reaction in the presence of 10 mM of each of L-glutamate and glycine. This resulted in two components in the binding data (high affinity and low affinity), which most likely reflect the open and closed states of the NMDA receptor-linked ion channel (47). In the present study, we utilized saturating concentrations (100 mM) of L-glutamate and glycine, in accord with a previous investigation in the rat (46), which resulted in a single population of high-affinity binding sites for [3H]MK-801 in control fetal guinea pig cerebral cortex. Interestingly, whereas Diaz-Granados et al. (12) reported an overall decrease in the number of NMDA receptors in the rat brain after chronic ethanol exposure, the percentage of high-affinity [3H]MK-801 binding sites was increased in all ethanol-treated groups. These data suggest the possibility that, in the period immediately following ethanol exposure, there is an upregulation of NMDA receptors in the cerebral cortex. Excitotoxic injury may lead to delayed cell loss and decreased receptor populations at later time points. In support of this idea, Thomas et al. (41) recently reported that 6-day-old rat pups exposed to a single high dose (6 g/kg body weight) of ethanol demonstrated impaired learning at postnatal day 40. This ethanol-induced behavioural deficit could be prevented by administration of MK-801 during the withdrawal period 21 h after the dose of
ethanol. This group proposed that ethanol withdrawal-related NMDA receptor-mediated excitotoxicity may be one consequence accounting for ethanol-induced damage to the developing brain. Indeed, upregulation of NMDA receptors or receptor subunits (15,16,43), increased functional sensitivity of the NMDA receptor (45), and increased sensitivity to NMDA receptor-mediated excitotoxicity (7,17,19) have all been demonstrated to occur after chronic ethanol exposure. For most binding sites of the traditional receptor–ligand complex type, the affinity for any particular ligand is determined by the association and dissociation kinetics of the ligand at the receptor complex. If such is the case for MK-801 binding sites, a decrease in the affinity would imply an alteration in the kinetics of the binding site–ligand interaction. Our binding experiments were carried out under equilibrium conditions. It will be necessary to examine the nonequilibrium binding characteristics of [3H]MK-801 to determine how chronic prenatal ethanol exposure alters the affinity of the NMDA receptor for this ligand. Additionally, the effects of chronic prenatal ethanol exposure on the sensitivity of the NMDA receptor to the coagonists (glutamate, glycine) requires a more extensive experimental protocol. In preliminary experiments (8), we have observed that chronic prenatal ethanol exposure produced an increase in the expression of the NMDA receptor 2C subunit in the near-term fetus. The impact of this change on cerebral cortical NMDA receptors, and the expression levels of other NDMA receptor subunit subtypes in our model, are currently being investigated. In conclusion, our study in the near-term fetal guinea pig demonstrates that chronic prenatal ethanol exposure, via maternal ethanol administration, decreases cerebral cortical weight with minimal fetal death. This cerebral cortical growth restriction is temporally associated with decreased affinity and increased number of MK-801 binding sites. Our findings are consistent with the suggestion that chronic prenatal ethanol exposure alters NMDA receptors in the developing cerebral cortex, which may increase the sensitivity of this brian region to excitotoxic cell death.
ACKNOWLEDGEMENTS
This study was supported by an operating grant from the Medical Research Council of Canada (Grant # MT-8073).
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Chronic Ethanol Exposure Alters MK-801 Binding Sites in the Cerebral Cortex of the Near-Term Fetal Guinea Pig J. CHIU,* J. F. BRIEN,* P. WU,† J. H. EUBANKS,‡ L. ZHANG‡ AND J. N. REYNOLDS* *Department of Pharmacology and Toxicology, Queen’s University, Ontario, Canada †Department of Pharmacology, University of Toronto, Toronto, Canada ‡Playfair Neuroscience Unit, Toronto Hospital Western Division, Toronto, Canada Received 19 February 1998; Accepted 20 July 1998 CHIU, J., J. F. BRIEN, P. WU, J. H. EUBANKS, L. ZHANG AND J. N. REYNOLDS. Chronic ethanol exposure alters MK-801 binding sties in the cerebral cortex of the near-term fetal guinea pig. ALCOHOL 17(3) 215–221, 1999.—The mechanism of ethanol central nervous system (CNS) teratogenesis, resulting from chronic maternal ingestion of high-dose ethanol during pregnancy, is not clearly understood. One of the target sites for ethanol-induced damage in the developing brain is the cerebral cortex. It has been proposed that chronic prenatal ethanol exposure alters NMDA receptors in the developing cerebral cortex. To test this hypothesis, timed pregnant guinea pigs were administered one of the following oral treatments throughout gestation: 4 g ethanol/kg maternal body weight/day; isocaloric sucrose/pair-feeding; water; or no treatment (ad lib). Near-term fetuses were studied at gestational day (GD) 63 (term, about GD 68). This ethanol regimen produced a maternal blood ethanol concentration of 66 6 4 mM (304 6 19 mg/dl) at 1 h after the daily dose on GD 58. The chronic ethanol regimen decreased near-term fetal body weight (12–26% decrease), brain weight (23% decrease), and cerebral cortical weight (21% decrease), compared with the isocaloric sucrose/pair-feeding, and combined water/ad lib experimental groups. Saturation analysis of near-term fetal cerebral cortical membranes using a [3H]MK-801 radioligand binding assay demonstrated a decreased affinity and increased number of MK-801 binding sites for the chronic ethanol regimen compared with the control treatments. These data support the suggestion that upregulation of NMDA receptors in the cerebral cortex after chronic prenatal ethanol exposure could lead to NMDA receptor-mediated excitotoxicity in this brain region. © 1999 Elsevier Science Inc. All rights reserved. Ethanol
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ment, including neuronal migration of cortical neurons (29), cell-cycle kinetics (31), and spine distribution of pyramidal cells (40). Conceivably, prenatal ethanol exposure can have deleterious effects on neurogenesis and synaptogenesis, thereby impairing neuronal function. Currently, there is no effective treatment for the neuronal defects induced by prenatal ethanol exposure, primarily because the mechanism of ethanol-induced damage of the developing CNS is not well understood. Recently, it was proposed that perturbation of synaptic transmission mediated by the N-methyl-D-aspartate (NMDA) receptor plays a key role in the mechanism of ethanol-induced damage of the developing CNS (36). The NMDA receptor is a subfamily of excitatory amino acid receptors of the L-gluta-
IT is well established that ethanol can injure the developing fetus. Chronic excessive ethanol (alcohol) consumption during pregnancy can lead to a specific pattern of birth defects called the fetal alcohol syndrome (FAS) (21). The primary features of FAS are growth restriction, craniofacial abnormalities, and central nervous system (CNS) dysfunction (10). Of all these defects, CNS dysfunction appears to be the most debilitating one, and its manifestations include motor incoordination, developmental delay, neurological abnormalities, and intellectual impairment. One of the target sites of ethanol in the developing brain is the cerebral cortex, which plays a key role in cognitive function and regulation of behaviour. Prenatal ethanol exposure can interfere with neuronal develop-
Requests for reprints should be addressed to Dr. J. N. Reynolds, Department of Pharmacology and Toxicology, Queen’s University, Kingston, Ontario K7L 3N6, Canada. Tel: (613) 533-6946; Fax: (613) 533-6412; E-mail: [email protected]
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mate pathway (32). It is a ligand-gated ion channel that has high permeability to calcium (26) and requires glycine as a coagonist (20,24). The NMDA receptor has many important roles during brain development; its activation promotes neuronal survival and differentiation (4), synaptogenesis (11), and formation of neuronal circuitry (35). Optimal activation of the L-glutamate–NMDA receptor system is considered to be essential for normal brain development; underactivation or overactivation of this system during development may lead to brain dysmorphology and dysfunction (26,36). Any substance perturbing NMDA receptor function, therefore, could interfere with developmental processes in the brain. In the hippocampus, another target site of ethanol-induced brain damage, chronic maternal ethanol administration decreased the number of NMDA-sensitive [3H]glutamate binding sites, but not their affinity, in the near-term fetal guinea pig (1). Similarly, prenatal ethanol exposure produced a decrease in NMDA-sensitive [3H]glutamate binding in the hippocampus of the adult rat (37). It has been reported that chronic prenatal ethanol exposure decreases NMDA-stimulated Ca21 entry in dissociated neurons obtained from neonatal rat frontal cortex (25), accompanied by decreased expression of specific subunit subtypes of the NMDA receptor complex (18,38). The present study was focused on testing the hypothesis that chronic prenatal ethanol exposure alters NMDA receptors in the developing cerebral cortex. The objective was to investigate, in the near-term fetal cerebral cortex, the effect of chronic prenatal ethanol exposure on NMDA receptor number and affinity. The guinea pig was selected as the experimental animal because its brain growth spurt occurs prenatally during the third-trimester-equivalent of gestation, which is more similar to the human situation compared with other rodent species, including the rat (13). The ethanol regimen used in this study has been shown previously to produce damage to the developing CNS (1,3,6,23). METHOD
Materials (1)-[3H]MK-801 (23.9 Ci/mmol) was supplied by DuPont New England Nuclear (Boston, MA). (1)-MK-801 maleate was obtained from Tocris Cookson (St. Louis, MO). All other chemicals were at least reagent grade, and were purchased from Sigma Chemical Co. or Fischer Scientific. Tap water was used to prepare the chemical cocktail for gas–liquid chromatographic (GLC) analysis of maternal blood ethanol concentration, because it has been found to be the most reliable source of water containing no GLC-interfering volatile organic compounds. All other aqueous solutions and buffers were prepared using deionized water obtained from a Barnstead Nanopure® system. Experimental Animals and Chronic Treatment Regimens Nulliparous Dunkin–Hartley strain guinea pigs were purchased from Charles River Canada Inc. (St. Constant, Quebec, Canada). All animals were kept in a room at an ambient temperature of 238C, with a 12-h light/12-h dark cycle with lights on at 0700 h. Female guinea pigs (500–600 g body weight) were bred with male guinea pigs (body weight . 700 g) of the same strain using an established procedure (14). The last day of full vaginal membrane opening was taken as gestational day (GD) 0. On GD 1, the pregnant animals were separated and housed singly in stainless steel wire cages. The
pregnant guinea pigs were assigned randomly to one of four experimental groups, comprising no treatment with ad lib acces to pellet food (Purina Guinea Pig Chow 5025®) and water, or one of the following three chronic treatments from GD 2 to GD 62 via intubation into the oral cavity: 4 g ethanol/kg maternal body weight/day with ad lib access to pellet food and water; isocaloric sucrose and pair-feeding to a particular ethanol-treated pregnant animal with ad lib access to water; or water with ad lib access to pellet food and water. Pair-fed animals were matched with an ethanol-treated animal, and given the same amounts of lab chow consumed by the ethanol-treated animal on each day of drug administration. This protocol ensured that ethanol-treated and isocaloric sucrose pair-fed animals received the same amount of calories on any given day during the dosing regimen. The ethanol (30% v/v) and sucrose (42% w/v) solutions were prepared with tap water. The ethanol, isocaloric sucrose, and water treatment regimens were administered as two equally divided volumes, given 2 h apart between 1100 and 1500 h. The pregnant animals were weighed and their vaginal membrane status was monitored daily. All animals were cared for according to the principles and guidelines of the Canadian Council on Animal Care. The experimental protocol was approved by the Queen’s University Animal Care Committee. Maternal Blood Ethanol Concentration Determination On GD 58, a maternal blood sample was collected from an ear vein at 1 h after the second divided volume of the daily dose of ethanol for the determination of ethanol concentration using an established GLC method involving head-space gas analysis (39). The blood ethanol concentration was determined in duplicate. Tissue Collection On GD 63, individual pregnant guinea pigs received no treatment and were killed by decapitation. The litter was delivered by cesarean section, and each fetus was decapitated. The fetal brain was excised, and the cerebral cortex was dissected. The body, brain, and cerebral cortical weights, and gender of each fetus were determined. The cerebral cortex was frozen immediately in liquid nitrogen, and was stored at 2708C until analyzed. Crude Plasma Membrane Preparation for Radioligand Binding Assay Each frozen cerebral cortex was thawed and homogenized in 20 ml/g tissue of buffer A (300 mM sucrose, 10 mM Tris, 2 mM EDTA, pH 7.3). The plasma membrane was obtained by differential centrifugation. In brief, the homogenate was centrifuged at 1000 3 g for 20 min at 48C, after which the supernatant was collected and centrifuged at 16,000 3 g for 1 h at 48C. The resulting pellet was collected and resuspended in 3 ml of buffer A. This membrane preparation was stored at 2708C. On the day of experimentation, an aliquot of the membrane suspension (8–10 mg protein) was thawed, diluted in 0.5 ml of buffer B (20 mM HEPES, 1 mM EDTA, pH 7.0), and incubated at 378C for 30 min. Following incubation, the suspension was centrifuged at 48C for 10 min at 13,000 3 g. The pellet was washed four times by resuspending it in 1 ml of buffer B and centrifuging at 48C for 5 min at 13,000 3 g. The final pellet was resuspended in buffer B to obtain a final crude plasma membrane preparation containing 2–4 mg membrane protein/ml.
CHRONIC ETHANOL EXPOSURE ALTERS MK-801 BINDING [3H]MK-801 Binding Assay An incubation mixture (final volume 5 0.5 ml) containing 50 ml of the final crude plasma membrane preparation (100– 200 mg protein) was incubated with one of nine different concentrations of [3H]MK-801 ranging from 0.2 to 45 nM. The binding assay was performed in the presence of 100 mM L-glutamate and 100 mM glycine. Preliminary experiments performed on control fetal cerebral cortex using lower concentrations (10 mM of each) of glutamate and glycine resulted in binding to two sites; one with high affinity and one with low affinity for [3H]MK-801 (data not shown). Higher concentrations (100 mM) of these two coagonists eliminated the lowaffinity component. Comparison of one- and two-site fits to the data using the F-test (Prism, Graphpad Software, Inc., San Diego, CA) indicated that a two-site model did not fit the data significantly better than a one-site model (p . 0.05). The higher concentrations of glutamate and glycine were therefore used for all subsequent experiments, and experimental data were fit to a one-site binding equation. Nonspecific binding was determined by incubation with 10 mM nonradioactive MK-801. The binding assay was conducted in duplicate at 378C for 3 h. Bound [3H]MK-801 radioactivity was determined by rapid vacuum filtration onto Whatman GF/B glass fibre filters presoaked with ice-cold buffer B. The filters were rinsed immediately with two 5-ml aliquots of ice-cold buffer B, and then placed in a scintillation vial containing 5 ml of scintillation cocktail followed by incubation in the dark at room temperature for 15 h. The radioactivity retained on the filter was quantified by liquid scintillation spectrometry using a Beckman LS3800® scintillation counter. The concentration of protein in the membrane preparation was determined using a protein-dye binding assay (5). Data Analysis The number (Bmax) and affinity (Kd) of the [3H]MK-801 binding sites of individual fetuses were determined from their individual saturation isotherm data using nonlinear regression analysis (Prism, Graphpad Software Inc.). The data are presented as group means 6 SEM, and were analysed by randomized-design one-way ANOVA followed by the Student–Newman–Keuls test for multiple comparisons (Prism, Graphpad Software Inc.). RESULTS
Maternal Blood Ethanol Concentration The chronic oral regimen of 4 g ethanol/kg maternal body weight/day produced a maternal blood ethanol concentration of 66 6 4 mM (304 6 19 mg/dl; n 5 10) at 1 h after the administration of the second divided volume of the daily ethanol dose on GD 58. A previous study of ethanol disposition in the near-term maternal–fetal unit of the pregnant guinea pig has demonstrated that the maternal and fetal blood ethanol concentrations are of similar magnitude and appear to be maximal at about 1 h after oral administration of the last equally divided volume of 4 g ethanol/kg maternal body weight (9). The maternal blood ethanol concentration of the present study is comparable with the data of our other investigations in the guinea pig, in which this chronic ethanol regimen produced CNS injury (1,3,6,23). Pregnancy Outcome Data Daily handling and administration of water had no effect on any of the parameters measured in this study. For this rea-
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son, the water treatment and ad lib groups were combined for the purposes of data analysis. There was no maternal lethality for the ethanol, isocaloric sucrose/pair-feeding, water and ad lib groups; however, there was one incident of embryonic reabsorption in each of the ethanol and water experimental groups. The litter sizes of the pregnant guinea pigs at GD 63 in the ethanol, isocaloric sucrose/pair-feeding, and water/ad lib groups were 3 6 0.4 (n 5 8 litters); 3.4 6 0.3 (n 5 8 litters), 3.4 6 0.3 (n 5 12 litters), respectively, and there was no difference among the three experimental groups. The total numbers of male and female offspring for the ethanol, sucrose, and water/ad lib groups were, respectively, 14 and 10 (n 5 8 litters), 13 and 14 (n 5 8 litters), and 19 and 22 (n 5 12 litters). There was no difference in the percentage of females and percentage of males per litter among the ethanol, sucrose, and water/ad lib groups: ethanol group, 62 6 7% for male, 39 6 7% for female; sucrose group, 45 6 11% for male, 55 6 11% for female; water/ad lib group, 50 6 8% for male, 50 6 8% for female. Effects of Ethanol on Fetal Body, Brain, and Cerebral Cortical Weights Both chronic ethanol and isocaloric sucrose/pair-feeding treatments decreased fetal body weight (Fig. 1A). Ethanol treatment produced a 12% decrease (p , 0.01) compared with sucrose treatment, and a 26% decrease (p , 0.001) compared with the combined water treatment/ad lib group. Sucrose treatment decreased fetal body weight by 16% (p , 0.01) compared with the water treatment/ad lib group. Chronic ethanol treatment decreased brain weight by 23% (p , 0.001) (Fig. 1B) and cerebral cortical weight by 21% (p , 0.001) (Fig. 1C) compared with the other experimental groups. There was no difference in the brain and cerebral cortical weight data between the sucrose and water treatment/ad lib groups. [3H]MK-801 Binding in the Fetal Cerebral Cortex MK-801 binding site number and affinity in the near-term fetal cerebral cortex were determined using saturation isotherm analysis with [3H]MK-801 as the radioligand. Saturation binding data for the three treatment groups, and a representative Scatchard plot from each of the experimental groups, are shown in Figure 2. The Bmax of MK-801 binding sites in the cerebral cortex was increased by chronic prenatal ethanol exposure (Figs. 2A, 3A) compared to the isocaloric sucrose and water/ad lib experimental groups. The Kd of the MK-801 binding sites for the ethanol group was increased by 57% (p , 0.001) compared with the other experimental groups. DISCUSSION
The regimen of 4 g ethanol/kg maternal body weight/day administered throughout gestation produced body, whole brain, and cerebral cortical growth restriction in the nearterm fetal guinea pig. Isocaloric sucrose/pair-feeding treatment only produced body growth restriction and did not affect whole brain and cerebral cortical weight, manifesting the “brain sparing effect” associated with undernutrition (33,34). These data demonstrate that undernutrition is not the mechanism underlying ethanol-induced alterations in MK-801 binding or weight of the cerebral cortex of the guinea pig; ethanol per se can produce these effects in this brain region. In the present study, [3H]MK-801 was utilized to examine the NMDA receptors in the near-term fetal guinea pig cere-
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CHIU ET AL. bral cortex. MK-801 is a selective, noncompetitive, reversible antagonist of the NMDA receptor. Because its binding site is in the ion channel (22,26), the value of maximum specific binding of [3H]MK-801 under equilibrium conditions is generally considered to be equivalent to the number of NMDA receptors. Channel opening is essential for the high-affinity specific binding of [3H]MK-801; therefore, saturating concentrations of L-glutamate and glycine were used in our binding assay to ensure maximum NMDA receptor activation. In the near-term fetal cerebral cortex, chronic maternal ethanol administration decreased the affinity of [3H]MK-801 binding sites and increased their number. Given the specific NMDA ion channel binding properties of nanomolar concentrations of MK-801, the data indicate that prenatal ethanol exposure increases the number of NMDA receptors in the near-term fetal guinea pig cerebral cortex. In contrast, chronic maternal ethanol administration decreased the number, but not the affinity, of NMDA-sensitive [3H]glutamate binding sites in the hippocampus of the near-term fetal guinea pig (1). Collectively, the data from the two studies demonstrate that chronic prenatal ethanol exposure alters NMDA receptors in both the cerebral cortex and hippocampus, but apparently by different mechanisms. Decreased number of NMDA receptors in the hippocampus after chronic prenatal ethanol exposure has been a consistent finding from several different laboratories (1,37,44). This effect of prenatal ethanol exposure on hippocampal NMDA receptors persists into the postnatal period (37), and may be a consequence of neuronal cell loss in the CA1 region of the hippocampus (30). The effects of chronic prenatal ethanol exposure on cerebral cortical NMDA receptors are not clearly understood. As the present manuscript was being prepared, another study was published (12) demonstrating that prenatal and early postnatal ethanol exposure in the rat decreased the number of [3H]MK-801 binding in a combined cerebral cortical/hippocampal preparation. This same group has also published data suggesting that chronic prenatal ethanol exposure decreases the expression of NR 2B subunit protein in the rat cerebral cortex, but has no effect on NR 1 subunit expression (18,38). The reason for the apparent discrepancy between the [3H]MK-801 binding data of our study (decreased affinity, increased number) and the recently published investigation of Diaz-Granados et al. (decreased number) (12), is uncertain. However, there are some significant methodological differences that make direct comparisons between the two studies very difficult. First, the developmental stage at which the animals were examined differed between the two studies. Whereas Diaz-Granados et al. (12) examined [3H]MK-801 binding in a combined hippocampal/cerebral cortical membrane preparation obtained from 20–23-day-old rats, we
FIG. 1. Body and brain weight data for near-term fetal guinea pigs. (A) Body weight of near-term fetuses of pregnant guinea pigs that received 4 g ethanol/kg maternal body weight/day (n 5 24), isocaloric sucrose/pair-feeding (n 5 27), or water/no treatment (ad lib) (n 5 41) from GD 2 to GD 62. **Indicates statistical difference (p , 0.05)
between ethanol and each of the other two experimental groups. *Indicates statistical difference (p , 0.01) between sucrose and the water/ad lib (AL) group. (B) Whole brain weight of near-term fetuses of pregnant guinea pigs of the three experimental groups described in (A) (ethanol, n 5 24; sucrose, n 5 27; water/ad lib, n 5 41). *Indicates statistical difference (p , 0.001) between ethanol and each of the other two experimental groups. (C) Cerebral cortical weights of near-term fetuses of pregnant guinea pigs of the three experimental groups described in (A) (ethanol, n 5 9; sucrose, n 5 11, water/ad lib, n 5 13). *Indicates statistical difference (p , 0.001) between ethanol and each of the other two experimental groups.
CHRONIC ETHANOL EXPOSURE ALTERS MK-801 BINDING
FIG. 2. [3H]MK-801 binding to cerebral cortical membranes prepared from near-term fetal guinea pigs. Animals were randomly selected from three different litters. (A) Saturation isotherms for [3H]MK-801 (0.2–45 nM) binding to cerebral cortical membranes in the presence of saturating concentrations of L-glutamate (100 mM) and glycine (100 mM). The best fit to the experimental data was used to determine the Kd and Bmax for [3H]MK-801 binding for ethanol (solid line), isocaloric sucrose pair-fed (hatched line), and water/ad lib groups (dashed line). (B) Representative Scatchard plots of [3H]MK-801 binding for each of the three experimental groups. A clear change in the slope of the ethanol-treated animal (solid line) and difference in the x-intercept reflects the increased Kd and Bmax in this group compared to both isocaloric sucrose pair-fed (hatched line) and water/ad lib animals (dashed line).
tested a cerebral cortical membrane preparation from the near-term fetal guinea pig. The ontogenic profile of NMDA receptor expression in the hippocampus and cerebral cortex differ dramatically between the rat and the guinea pig. In the rat, NMDA receptor expression in the hippocampus and cerebral cortex undergoes a transient upregulation that begins
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FIG. 3. Characteristics of [3H]MK-801 binding sites in the cerebral cortex of the near-term fetal guinea pig. (A) Bmax of the MK-801 binding sites in the cerebral cortex of near-term fetuses of the three experimental groups described in Fig. 1A (ethanol, n 5 9; isocaloric sucrose/pair-feeding, n 5 5; water/ad lib, n 5 6). The data are the mean 6 SEM of the Bmax values obtained from the saturation isotherms of the individual fetuses. Ethanol increased the Bmax values for [3H]MK-801 binding to near-term fetal guinea pig cerebral cortex. *Indicates statistical difference (p , 0.05) between ethanol and each of the other two experimental groups. (B) Kd of the specific binding of [3H]MK-801 to NMDA receptors in the cerebral cortex of nearterm fetuses of the three experimental groups. The data are the mean 6 SEM of the Kd values obtained from the saturation isotherms of the individual fetuses. *Indicates statistical difference (p , 0.001) between ethanol and each of the other two experimental groups.
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during the first postnatal week and peaks between postnatal days 9 and 15 (27,28,42). Thereafter, NMDA receptor density in these brain regions declines rapidly, reaching adult levels by postnatal days 20–28. In contrast, the transient increase in the density of NMDA receptors in the guinea pig hippocampus beings prenatally between GD 50 and GD 62 (2). NMDA receptor density in the guinea pig hippocampus peaks in the near-term fetus (around GD 62) and remains elevated until at least the second postnatal week, and thereafter declines towards adult levels. Second, in the present study we obtained cerebral cortical tissue from near-term fetal guinea pigs 24 h after the last dose of ethanol. Diaz-Granados et al. (12) used a combined hippocampal/cerebral cortical membrane preparation obtained from 20–23-day-old rats 11–14 days after the last dose of ethanol. This suggests the possibility that ethanol withdrawal may be an important determinant of ethanol-induced damage to the developing brain. Third, the assay conditions in which [3H]MK-801 binding was examined were different between the two studies. Diaz-Granados et al. (12) utilized a combined hippocampal/cerebral cortical membrane preparation, and carried out the binding reaction in the presence of 10 mM of each of L-glutamate and glycine. This resulted in two components in the binding data (high affinity and low affinity), which most likely reflect the open and closed states of the NMDA receptor-linked ion channel (47). In the present study, we utilized saturating concentrations (100 mM) of L-glutamate and glycine, in accord with a previous investigation in the rat (46), which resulted in a single population of high-affinity binding sites for [3H]MK-801 in control fetal guinea pig cerebral cortex. Interestingly, whereas Diaz-Granados et al. (12) reported an overall decrease in the number of NMDA receptors in the rat brain after chronic ethanol exposure, the percentage of high-affinity [3H]MK-801 binding sites was increased in all ethanol-treated groups. These data suggest the possibility that, in the period immediately following ethanol exposure, there is an upregulation of NMDA receptors in the cerebral cortex. Excitotoxic injury may lead to delayed cell loss and decreased receptor populations at later time points. In support of this idea, Thomas et al. (41) recently reported that 6-day-old rat pups exposed to a single high dose (6 g/kg body weight) of ethanol demonstrated impaired learning at postnatal day 40. This ethanol-induced behavioural deficit could be prevented by administration of MK-801 during the withdrawal period 21 h after the dose of
ethanol. This group proposed that ethanol withdrawal-related NMDA receptor-mediated excitotoxicity may be one consequence accounting for ethanol-induced damage to the developing brain. Indeed, upregulation of NMDA receptors or receptor subunits (15,16,43), increased functional sensitivity of the NMDA receptor (45), and increased sensitivity to NMDA receptor-mediated excitotoxicity (7,17,19) have all been demonstrated to occur after chronic ethanol exposure. For most binding sites of the traditional receptor–ligand complex type, the affinity for any particular ligand is determined by the association and dissociation kinetics of the ligand at the receptor complex. If such is the case for MK-801 binding sites, a decrease in the affinity would imply an alteration in the kinetics of the binding site–ligand interaction. Our binding experiments were carried out under equilibrium conditions. It will be necessary to examine the nonequilibrium binding characteristics of [3H]MK-801 to determine how chronic prenatal ethanol exposure alters the affinity of the NMDA receptor for this ligand. Additionally, the effects of chronic prenatal ethanol exposure on the sensitivity of the NMDA receptor to the coagonists (glutamate, glycine) requires a more extensive experimental protocol. In preliminary experiments (8), we have observed that chronic prenatal ethanol exposure produced an increase in the expression of the NMDA receptor 2C subunit in the near-term fetus. The impact of this change on cerebral cortical NMDA receptors, and the expression levels of other NDMA receptor subunit subtypes in our model, are currently being investigated. In conclusion, our study in the near-term fetal guinea pig demonstrates that chronic prenatal ethanol exposure, via maternal ethanol administration, decreases cerebral cortical weight with minimal fetal death. This cerebral cortical growth restriction is temporally associated with decreased affinity and increased number of MK-801 binding sites. Our findings are consistent with the suggestion that chronic prenatal ethanol exposure alters NMDA receptors in the developing cerebral cortex, which may increase the sensitivity of this brian region to excitotoxic cell death.
ACKNOWLEDGEMENTS
This study was supported by an operating grant from the Medical Research Council of Canada (Grant # MT-8073).
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