Dose-Dependent Effects of Acute In Vivo Ethanol Exposure on Extracellular Glutamate Concentration in the Cerebral Cortex of the Near-Term Fetal Sheep

The cerebral cortex is a target site of ethanol teratogenesis. l -Glutamate is a major excitatory neurotransmitter that plays an important neurotrophic role in brain development. It has been proposed that optimal function of the glutamate neuronal system is required for normal brain development; overactivation could lead to excitotoxic-induced neuronal injury, whereas underactivation could delay/restrict brain development. The objective of this study was to test the hypothesis that acute in vivo ethanol exposure alters basal glutamate release in the fetal cerebral cortex. The experimental approach involved measuring fetal cortical extracellular glutamate concentration using the technique of in vivo microdialysis. Near-term fetal sheep were chronically instrumented with a microdialysis probe placed in the parasagittal cortex. At 124 ± 3 days of gestation, the effects of maternal intravenous infusion of 2 g or 4 g ethanol/kg maternal body weight or an equivalent volume of saline, given as four equally divided doses over 5 hr, on fetal cerebral cortical extracellular glutamate concentration were determined. None of the three treatment regimens produced fetal or maternal demise during the time course of the study. There was an ethanol dose-dependent increase, p = 0.005, in extracellular glutamate concentration in the fetal cerebral cortex. This increase was paroxysmal in nature and was not directly related to the fetal blood ethanol concentration. In view of the proposed role for glutamate in neuronal development, this apparent ethanol-induced increase in glutamate release may be important in the pathogenesis of ethanol teratogenesis involving the cerebral cortex.     

Ethanol Inhibits NMDA Receptor-Mediated Excitotoxicity in Rat Primary Neuronal Cultures

Excessive or prolonged stimulation of, N -methyl- D -aspartate (NMDA) receptors appears to play an important role in many neurodegenerative processes in brain through a process known as excitotoxicity. This study examined the effects of ethanol on NMDA receptormediated excitotoxicity in primary neuronal cultures obtained from embryonic rat whole brain. Neurotoxicity was quantitated by measuring the amount of lactate dehydrogenase released into the media during a 20-hr time period following NMDA washout. Exposure of 12-to 14-day-old cultures to NMDA in Mg²⁺-free HEPES buffer (pH 7.4) for a 25-min period resulted in a concentration-dependent toxicity (EC₅₀= 54 μM). Time-course experiments showed that exposure to NMDA for as little as 5 min was excitotoxic and reached a plateau after a 20-min exposure period. Preincubation of the cultures with ethanol (25 to 200 mm) resulted in a concentration-dependent inhibition of NMDA-mediated toxicity with approximately 38% inhibition produced by 25 mm ethanol and essentially complete inhibition at 200 mm ethanol (IC₅₀= 60 mm). Increasing the glycine concentration to 100 μM did not potentiate NMDA neurotoxicity or antagonize the neuroprotective effect of ethanol. NMDA-Mediated excitotoxicity was reduced by approximately 50% by the glycine antagonist 7-chlorokynurenate (50 μM). Ethanol (50 mm) reduced NMDA neurotoxicity similar to 7-chlorokynurenate, and the two together produced greater inhibition than either alone. These results show that intoxicating concentrations of ethanol can potently inhibit NMDA receptor-mediated excitotoxicity and may have important implications in terms of ethanols interactions with brain trauma, ischemia, and other neuropathologies associated with NMDA receptor-mediated neurotoxicity.     

Catalase Mediates Acetaldehyde Formation from Ethanol in Fetal and Neonatal Rat Brain

Fetal ethanol (E) exposure has well documented deleterious effects on brain development, yet it is uncertain if the neurotoxicity of maternal E consumption is generated by E itself, by its primary metabolite acetaldehyde (AcHO), or both. The current studies present evidence that homogenates of immature rat brains can generate AcHO via a catalase (CAT)-mediated reaction and that AcHO may be produced in vivo by this system. Homogenates of day 19 fetal rat brain were incubated with E (50 mM). When incubated with CAT inhibitors (sodium azide or 3-aminotriazole), AcHO formation was blocked, whereas neither the alcohol dehydrogenase inhibitor, 4-methylpyratole, nor P-450 inhibitors decreased AcHO production. Three hours after one oral dose of E (4 g/kg) to a pregnant dam (gestation day 19), AcHO levels in fetal brain increased to 14.28 ± 1.82 nM/g tissue. Baseline CAT activity in day 19 fetal brains was 4.5 times adult values ( p < 0.05). Western blot analysis determined that CAT protein level in the day 19 fetal brain exceeded that in adult brain by 2.5 times. One hour after a single dose of E, CAT activity in day 19 fetal brain increased by 8.2 units/mg protein. In 5-day-old neonatal brains during the "third trimester" brain growth spurt, baseline CAT activity was twice the adult values ( p < 0.05) and a 2-day in vivo E regimen increased AcHO levels to four times the control values, with a concomitant 1.7-fold increase in CAT activity. This was prevented by administration of a CAT inhibitor (3-amino-1,2,4-triazole). Immunohistochemical staining of neonatal brains exposed to E illustrated the presence of acetaldehyde-protein adducts. We conclude that AcHO is likely produced in rat fetal and neonatal brain via CAT-mediated oxidation of E. This phenomenon may be an important factor in the neurotoxic effects of in utero E exposure.     

Effect of Repeated Ethanol Withdrawal on Glutamate Microdialysate in the Hippocampus

BACKGROUND: Our previous studies, which identified that ethanol withdrawal is associated with increases in glutamate microdialysate in the nucleus accumbens and reaches a maximum at 12 hr, have now been extended in order to assess whether repeated cycles of chronic ethanol intoxication followed by 12 hr withdrawal periods on three occasions alters glutamate release in the hippocampus of male rats. METHODS: In this study, the microdialysis technique has been used with the HPLC and electrochemical detection. RESULTS: During the first cycle of ethanol withdrawal, glutamate content increased significantly 8 hr after withdrawal (198.4% +/- 89.14%) by comparison with control rats. During the second period of ethanol withdrawal, 1 week after the initial withdrawal episode, glutamate microdialysate content increased significantly 10 hr after withdrawal, but to a much lower degree than in the first episode (179.08 +/- 25.68%), by comparison with control rats. During the third cycle of ethanol withdrawal, the concentration of glutamate in the hippocampus microdialysate did not significantly change at either of these time points except at 12 hr when glutamate was significantly decreased by comparison with control rats (52.09 +/- 14.38%). Apart from arginine, which was significantly decreased both at the cessation of alcoholization and during the 12 hr of the three withdrawal episodes, none of the other neurotransmitters assayed, aspartate, taurine, alanine, or GABA, showed any significant alteration. CONCLUSION: These results clearly indicate that elevated glutamate release during the first withdrawal episode is not paralleled in subsequent withdrawal episodes.     

Ethanol Reduces Expression of the Nerve Growth Factor Receptor, But Not Nerve Growth Factor Protein Levels in the Neonatal Rat Cerebellum

The cerebellum is especially vulnerable to ethanol's neurotoxic effects during development, and ethanol exposure during the brain growth spurt will deplete cerebellar neurons. The mechanisms underlying this neuronal cell loss remain elusive. Nerve growth factor (NGF) is a neurotrophin that promotes cell survival in various brain areas, and there is evidence that NGF may play a role in the developing cerebellum. This study examined whether ethanol exposure of the neonatal rat cerebellum altered the levels of either NGF or the expression of p75 and trkA, which are two components of the NGF receptor. Ethanol exposure had no effect on NGF levels in the neonatal cerebellum, as determined by an NGF-specific ELISA. Immunohistochemical labeling techniques indicated that both the p75 and trkA NGF receptors were expressed on Purkinje cell dendrites in the developing cerebellum, with posterior lobules expressing higher levels of p75 and trkA NGF receptor, compared with anterior lobules. Ethanol exposure of neonatal rats reduced the expression of both p75 and trkA NGF receptors on the Purkinje cell dendrites. These results suggest that ethanol could interfere with neurotrophic support of Purkinje cells by reducing the levels of available NGF receptor.     

In Utero Ethanol Exposure Elicits Oxidative Stress in the Rat Fetus

Prior studies in our laboratory have shown that exposure of cultured fetal rat hepatocytes to ethanol (E) blocks epidermal growth factor-dependent replication and that this is paralleled by cell membrane damage, mitochondrial dysfunction, membrane lipid peroxidation (LP), and enhanced generation of reactive oxygen species. These measures of E-mediated oxidative stress (OS) were mitigated by treatment with antioxidants, and cell replication could be normalized by maintaining cell glutathione (GSH) pools. We have now extended these studies to an in vivo model. Rats were administered E (4 g/kg, po) at 12-hr intervals on days 17 and 18 of gestation and killed on day 19,1 hr following a final dose of E (a total of 5 doses). Fetal and maternal brain and liver were assayed for signs of OS. The 2-day in utero E exposure increased membrane LP in fetal brain as evidenced by increased malondialdehyde (MDA) levels from 1.76 ± 0.12 se (nMol/mg protein) to 2.00 ± 0.08 (p < 0.05) and conjugated dienes from 0.230 ± 0.006 se (OD₂₃₃/mg lipid) to 0.282 ± 0.006 (p < 0.05). In fetal liver, MDA levels increased from 2.39 ± 0.08 se (nMol/mg protein) to 2.87 ± 0.08 (p < 0.05), whereas dienes differed significantly only between ad libitum controls and the E and pair-fed control groups (p < 0.05). E decreased GSH levels in fetal brain by 19%, from 19.88 ± 0.72 to 16.13 ± 1.06 (nMol/mg protein) (p < 0.05). A10% decrease in GSH was seen in fetal liver (p < 0.05). GSH in maternal brain was decreased by 44% from 47.29 ± 3.38 to 26.60 ± 2.29 (p < 0.05). Other E-related increases in these OS measures were not observed in maternal organs. E did not decrease α-tocopherol levels in fetal and maternal brain or in fetal liver (p < 0.05), whereas maternal liver α-tocopherol content was reduced by 31% (p < 0.05) by E treatment. It is concluded that maternal E consumption can induce an OS in fetal tissues that may contribute to the fetotoxic effects of E.     

Effect of Ethanol on the Release of Prostaglandins from Ovine Fetal Brain Stem during Gestation

Prostaglandins (PGs) have been implicated as mediators of the ethanol-induced suppression of ovine fetal breathing movements (FBM). The objectives of the present study were to determine the ontogeny of the in vitro efflux of PGE2 and 6-keto PGF1 alpha in ovine fetal brain stem during the second half of gestation and to determine the effect of in vitro ethanol exposure on the efflux of these PGs. Ovine fetal brain stem tissue was obtained at mean gestational ages of 80 days (n = 6), 105 days (n = 10), and 135 days (n = 16) by rapid excision following maternal euthanization. Tissue slices (400 microM thickness) were prepared from the lower pons-medulla region of the brain stem. After a 1-hr equilibration period in artificial cerebrospinal fluid, efflux of PGE2 and 6-keto PGF1 alpha in the brain stem was determined using the brain slice-superfusion method, and the PGE2 and 6-keto PGF1 alpha concentrations in the superfusate were determined by specific radioimmunoassay. The mean spontaneous efflux of PGE2 and 6-keto PGF1 alpha expressed as pmol PG/gram wet weight of tissue/5-min collection period was, respectively, 31.9 +/- 4.2 and 26.6 +/- 2.4 at 80 days, 38.3 +/- 5.2 and 29.6 +/- 2.2 at 105 days, and 57.4 +/- 3.1 and 27.1 +/- 1.1 at 135 days of gestation. In vitro exposure to 20, 40, and 80 mM ethanol did not affect PG efflux in the brain stem at 80 and 105 days of gestation. In vitro ethanol exposure decreased PGE2 and 6-keto PGF1 alpha efflux at 135 days of gestation to 36.8 +/- 5.3% and 41.6 +/- 4.3% of spontaneous efflux within 15 min, respectively; this effect of ethanol was not dose-dependent. The data do not support the hypothesis that ethanol increases PG efflux in the ovine fetal brain stem. In view of these findings and the data implicating PGs in the mechanism of ethanol-induced suppression of FBM, it is possible that ethanol acts at either central sites rostral to the brain stem (i.e., upstream CSF) or peripheral sites to increase the synthesis of PGs and their efflux into the systemic circulation, with subsequent transfer to the respiratory control region(s) of the brain stem.     

NMDA Receptor Binding in Adult Rat Brain after Several Chronic Ethanol Treatment Protocols

The amino acid L-glutamate is a major excitatory neurotransmitter that is involved in many CNS functions, including learning, memory, long-term potentiation, and synaptic plasticity. Acute exposures to ethanol (50 to 200 mM) have been shown to inhibit NMDA receptor responses, whereas chronic exposure to ethanol leads to adaptive supersensitivity thought to be involved in ethanol dependence and tolerance. To investigate the effects of chronic ethanol exposure on glutamate receptor density, we examined the binding of both NMDA and non-NMDA ligands in rat brain after several chronic ethanol treatment protocols using a number of different rat strains. No increases in the binding of [³H]MK-801, [³H]CGP 39653, or the polyamine specific competitive antagonist, [³H]ifenprodil, were seen after two well-used chronic ethanol treatments. These included the 2-week liquid diet developed by Frye et al. (J. Pharmacol. Exp. Ther. 216:306-314, 1981) and the 4-day binge treatment developed by Ma-jchrowicz (Psychopharmacologia 43:245-254, 1975). However, small increases in the binding of both the NMDA noncompetitive antagonist [³H]MK-801, as well as the competitive NMDA antagonist [³H]CGP 39653, were seen in select frontal brain regions after 3 weeks of the Walker-Freund chronic ethanol liquid diet When this chronic liquid diet treatment was extended to a period of 6 weeks, these increases in receptor binding were diminished to nonsignificant levels. The binding of the non-NMDA ligands [³H]AMPA and [³H]kainate were not significantly affected by either length of Walker-Freund liquid diet exposure. When rats were treated chronically with ethanol for 30 days using the paradigm developed by Tsukamoto et al. (Hepatology 5:224-232, 1985), small, but significant, increases in the binding of [³H]MK-801 were seen in the CA1 and dentate gyrus regions of the hippocampus. These studies indicate that robust increases in NMDA receptor binding do not occur with several chronic ethanol treatment protocols, and suggests that NMDA receptor supersensitivity during the development of tolerance and dependence to ethanol may not simply be due to changes in the density of NMDA receptors, but may involve other mechanisms.     

Fetal Exposure to Moderate Ethanol Doses: Heightened Operant Responsiveness Elicited by Ethanol-Related Reinforcers

Background: Prenatal exposure to moderate ethanol doses during late gestation modifies postnatal ethanol palatability and ingestion. The use of Pavlovian associative procedures has indicated that these prenatal experiences broaden the range of ethanol doses capable of supporting appetitive conditioning. Recently, a novel operant technique aimed at analyzing neonatal predisposition to gain access to ethanol has been developed. Experiment 1 tested the operant conditioning technique for developing rats described by Arias and colleagues (2007) and Bordner and colleagues (2008) . In Experiment 2, we analyzed changes in the disposition to gain access to ethanol as a result of moderate prenatal exposure to the drug. Methods: In Experiment 1, newborn pups were intraorally cannulated and placed in a supine position that allowed access to a touch‐sensitive sensor. Paired pups received an intraoral administration of a given reinforcer (milk or quinine) contingent upon physical contact with the sensor. Yoked controls received similar reinforcers only when Paired pups activated the circuit. In Experiment 2, natural reinforcers (water or milk) as well as ethanol (3% or 6% v/v) or an ethanol‐related reinforcer (sucrose compounded with quinine) were tested. In this experiment, pups had been exposed to water or ethanol (1 or 2 g/kg) during gestational days 17 to 20. Results: Experiment 1 confirmed previous results showing that 1‐day‐old pups rapidly learn an operant task to gain access to milk, but not to gain access to a bitter tastant. Experiment 2 showed that water and milk were highly reinforcing across prenatal treatments. Furthermore, general activity during training was not affected by prenatal exposure to ethanol. Most importantly, prenatal ethanol exposure facilitated conditioning when the reinforcer was 3% v/v ethanol or a psychophysical equivalent of ethanol’s gustatory properties (sucrose–quinine). Conclusions: The present results suggest that late prenatal experience with ethanol changes the predisposition of the newborn to gain access to ethanol‐related stimuli. In conjunction with prior literature, this study emphasizes the fact that intrauterine experience with ethanol not only augments ethanol’s palatability and ingestion, but also facilitates the acquisition of response–stimulus associations where the drug acts as an intraoral reinforcer.     

Ethanol Acutely Inhibits Ionotropic Glutamate Receptor‐Mediated Responses and Long‐Term Potentiation in the Developing CA1 Hippocampus

Background: Developmental ethanol (EtOH) exposure damages the hippocampus, causing long‐lasting alterations in learning and memory. Alterations in glutamatergic synaptic transmission and plasticity may play a role in the mechanism of action of EtOH. This signaling is fundamental for synaptogenesis, which occurs during the third trimester of human pregnancy (first 12 days of life in rats). Methods: Acute coronal brain slices were prepared from 7‐ to 9‐day‐old rats. Extracellular and patch‐clamp electrophysiological recording techniques were used to characterize the acute effects of EtOH on α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate receptor (AMPAR)‐ and N‐methyl‐d ‐aspartate receptor (NMDAR)‐mediated responses and long‐term potentiation (LTP) in the CA1 hippocampal region. Results: Ethanol (40 and 80 mM) inhibited AMPAR‐ and NMDAR‐mediated field excitatory postsynaptic potentials (fEPSPs). EtOH (80 mM) also reduced AMPAR‐mediated fEPSPs in the presence of an inhibitor of Ca²⁺ permeable AMPARs. The effect of 80 mM EtOH on NMDAR‐mediated fEPSPs was significantly greater in the presence of Mg²⁺. EtOH (80 mM) neither affected the paired‐pulse ratio of AMPAR‐mediated fEPSPs nor the presynaptic volley. The paired‐pulse ratio of AMPAR‐mediated excitatory postsynaptic currents was not affected either, and the amplitude of these currents was inhibited to a lesser extent than that of fEPSPs. EtOH (80 mM) inhibited LTP of AMPAR‐mediated fEPSPs. Conclusions: Acute EtOH exposure during the third‐trimester equivalent of human pregnancy inhibits hippocampal glutamatergic transmission and LTP induction, which could alter synapse refinement and ultimately contribute to the pathophysiology of fetal alcohol spectrum disorder.     

Ethanol Impairs Activation of Retinoic Acid Receptors in Cerebellar Granule Cells in a Rodent Model of Fetal Alcohol Spectrum Disorders

Background: Ethanol is the main addictive and neurotoxic constituent of alcohol. Ethanol exposure during embryonic development causes dysfunction of the central nervous system (CNS) and leads to fetal alcohol spectrum disorders. The cerebellum is one of the CNS regions that are particularly vulnerable to ethanol toxic effects. Retinoic acid (RA) is a physiologically active metabolite of vitamin A that is locally synthesized in the cerebellum. Studies have shown that RA is required for neuronal development, but it remains unknown if ethanol impairs RA signaling and thus induces neuronal malformations. In this study, we tested the hypothesis that ethanol impairs the expression and activation of RA receptors in cerebellum and in cerebellar granule cells. Methods: The cerebellum of ethanol unexposed and exposed pups was used to study the expression of retinoic acid receptors (RARs or RXRs) by immunohistochemistry and by Western blot analysis. We also studied the effect of ethanol on expression of RA receptors in the cerebellar granule cells. Activation of RA receptors (DNA‐binding activities) in response to high‐dose ethanol was determined by electrophoretic mobility shift and supershift assays. Results: Findings from these studies demonstrated that ethanol exposure reduced the expression of RARα/γ while it increased the expression of RXRα/γ in the cerebellum and in cerebellar granule neurons. Immuno‐histological studies further strengthened the expression pattern of RA receptors in response to ethanol. The DNA‐binding activity of RARs was reduced, while DNA‐binding activity of RXRs was increased in response to ethanol exposure. Conclusion: For the first time, our studies have demonstrated that high‐dose ethanol affects the expression and activation of RA receptors, which could impair the signaling events and induce harmful effects on the survival and differentiation of cerebellar granule cells. Taken together, these findings could provide insight into the treatment options for brain defects caused by excessive ethanol exposure, such as in Fetal Alcohol Spectrum Disorders.     

Acamprosate is neuroprotective against glutamate-induced excitotoxicity when enhanced by ethanol withdrawal in neocortical cultures of fetal rat brain

BACKGROUND: Acamprosate reduces relapse, and the drug may interact with glutamate transmission and with glutamate/NMDA receptors. Because these systems are implicated in excess calcium entry leading to alcohol withdrawal-induced neurotoxicity, we evaluated the effects of acamprosate on these parameters in neuronal cultures. METHODS: Primary cultures of neocortical neurons from fetal Sprague-Dawley rats were maintained either in normal medium or in medium containing 100 mM ethanol for 4 days. After this time, cultures were challenged with glutamate for 10 min and then returned to medium (all in the absence of ethanol). 45Ca2+ uptake was measured during the challenge, and glutamate-induced toxicity was assessed after 20 hr. The effects of acamprosate present during the glutamate challenge were measured on both parameters. RESULTS: In controls, acamprosate did not significantly affect glutamate-induced neurotoxicity but produced a significant inhibition of calcium entry. The NMDA receptor antagonists dizocilpine and d-amino-phosphonovalerate (D-APV) inhibited both glutamate neurotoxicity and calcium entry. In cultures previously exposed to ethanol, glutamate-induced neurotoxicity and calcium entry were both significantly enhanced. Dizocilpine reduced both these parameters to unstimulated control values, and D-APV reduced both calcium entry and neurotoxicity with the same relation that we obtained in control cultures. In the ethanol-withdrawn cultures, acamprosate reduced both the enhanced glutamate-induced calcium entry and the enhanced neurotoxicity in a concentration-dependent manner. Acamprosate also significantly reduced calcium entry caused by 80 mM K+ in control and ethanol-exposed cultures. CONCLUSIONS: Acamprosate had protective effects against glutamate-induced neurotoxicity only in ethanol-withdrawn cultures. The neuroprotective effects of the drug did not correlate with its effects on calcium entry, making it unlikely that acamprosate directly affects NMDA receptors via the glutamate binding site or the receptor-operated calcium channel. The results are, however, compatible with other inhibitory effects on NMDA receptor function.     

Similar Effects of Ethanol and tert-Butanol on Amino Acid Concentrations in Rat Serum and Liver

Both metabolic and nonmetabolic mechanisms have been proposed to the plasma amino acid decreasing effect of an acute ethanol load. We used tert-butanol, an alcohol that is only minimally metabolized, as a tool to explain the mechanism behind the amino acid decreasing effect of ethanol. Acute administration of tert-butanol was found to exert a decreasing effect on rat serum amino acid concentrations similar to that of ethanol, indicating that the mechanism of the amino acid decreasing effect of ethanol is primarily due to ethanol itself and not to its oxidation. Ethanol and tert-butanol also had similar effects on liver amino acid concentrations, including an increase in the glycine concentration and decreases in the concentrations of glutamate, alanine, leucine, and tyrosine.     

Effects of In Vitro Ethanol and Fetal Ethanol Exposure on Glutathione Stimulation of N-Methyl-D-Aspartate Receptor Function

The present studies investigated the effects of glutathione (GSH; γ-glutamylcysteinylglycine) and its oxidized form (GSSG) on neuronal N -methyl-D-aspartate (NMDA) receptor activation in both acute and chronic preparations of ethanol exposure. It was demonstrated using fura-2-loaded dissociated brain cells from newborn rat pups that both GSH and GSSG (0–4 mM) produced concentration-dependent increases in intracellular calcium similar to those produced by NMDA and other agonists of the NMDA receptor. GSH-stimulated calcium entry was not inhibited by low intoxicating concentrations of ethanol, which contrasts with ethanol's typical inhibitory effect on NMDA-stimulated receptor activation. Behavioral studies in adult rats demonstrated that ethanol-induced sleep times were significantly decreased when 10 μl of GSSG (20 mM) were administered intracere-broventricularly approximately 5 min before an intraperitoneal injection of 20% (w/v) ethanol (3 g/kg). These findings suggest that the less potent effect of ethanol on GSH-stimulated calcium entry as well as the reduction in ethanol-induced sleep times may be related to the presence of glycine in the peptide. The glycine found in GSH may activate the glycine site and block or reduce ethanol's action on this site. It appears that although GSH may play an important role in the activation of the NMDA receptor, this action does not involve a process that is sensitive to acute ethanol exposure. In contrast, when rat pups were chronically exposed to ethanol via prenatal exposure before the fura-2 preparation, increases in NMDA- and GSH-stimulated calcium entry were significantly decreased relative to those in pair-fed and ad libitum-fed controls. Thus, chronic in utero exposure to ethanol may alter the NMDA-receptor complex, such that calcium entry mediated by NMDA or GSH activation is significantly reduced.