Maternal Blood Ethanol Concentration Research Articles

Toxicological assessments of rats exposed prenatally to inhaled vapors of gasoline and gasoline–ethanol blends

The primary alternative to petroleum-based fuels is ethanol, which may be blended with gasoline in the United States at concentrations up to 15% for most automobiles. Efforts to increase the amount of ethanol in gasoline have prompted concerns about the potential toxicity of inhaled ethanol vapors from these fuels. The well-known sensitivity of the developing nervous and immune systems to ingested ethanol and the lack of information about the neurodevelopmental toxicity of ethanol-blended fuels prompted the present work. Pregnant Long–Evans rats were exposed for 6.5h/day on days 9–20 of gestation to clean air or vapors of gasoline containing no ethanol (E0) or gasoline blended with 15% ethanol (E15) or 85% ethanol (E85) at nominal concentrations of 3000, 6000, or 9000ppm. Estimated maternal peak blood ethanol concentrations were less than 5mg/dL for all exposures. No overt toxicity in the dams was observed, although pregnant dams exposed to 9000ppm of E0 or E85 gained more weight per gram of food consumed during the 12days of exposure than did controls. Fuel vapors did not affect litter size or weight, or postnatal weight gain in the offspring. Tests of motor activity and a functional observational battery (FOB) administered to the offspring between post-natal day (PND) 27–29 and PND 56–63 revealed an increase in vertical activity counts in the 3000- and 9000-ppm groups in the E85 experiment on PND 63 and a few small changes in sensorimotor responses in the FOB that were not monotonically related to exposure concentration in any experiment. Neither cell-mediated nor humoral immunity were affected in a concentration-related manner by exposure to any of the vapors in 6-week-old male or female offspring. Systematic concentration-related differences in systolic blood pressure were not observed in rats tested at 3 and 6months of age in any experiment. No systematic differences were observed in serum glucose or glycated hemoglobin A1c (a marker of long-term glucose homeostasis). These observations suggest a LOEL of 3000ppm of E85 for vertical activity, LOELs of 9000ppm of E0 and E85 for maternal food consumption, and NOELs of 9000ppm for the other endpoints reported here. The ethanol content of the vapors did not consistently alter the pattern of behavioral, immunological, or physiological responses to the fuel vapors. The concentrations of the vapors used here exceed by 4–6 orders of magnitude typical exposure levels encountered by the public.

Chronic prenatal ethanol exposure alters expression of central and peripheral insulin signaling molecules in adult guinea pig offspring

Maternal ethanol consumption during pregnancy can produce a range of teratogenic outcomes in offspring. The mechanism of ethanol teratogenicity is multi-faceted, but may involve alterations in insulin and insulin-like growth factor (IGF) signaling pathways. These pathways are not only important for metabolism, but are also critically involved in neuronal survival and plasticity, and they can be altered by chronic prenatal ethanol exposure (CPEE). The objective of this study was to test the hypothesis that CPEE alters expression of insulin and IGF signaling molecules in the prefrontal cortex and liver of adult guinea pig offspring. Pregnant Dunkin-Hartley-strain guinea pigs received ethanol (4 g/kg maternal body weight/day) or isocaloric-sucrose/pair-feeding (nutritional control) throughout gestation. Fasting blood glucose concentration was measured in male and female offspring at postnatal day 150–200, followed by euthanasia, collection of prefrontal cortex and liver, and RNA extraction. IGF-1, IGF-1 receptor (IGF-1R), IGF-2, IGF-2 receptor (IGF-2R), insulin receptor substrate (IRS)-1, IRS-2, and insulin receptor (INSR) mRNA expression levels were measured in tissues using quantitative real-time PCR. The mean maternal blood ethanol concentration was 281 ± 15 mg/dL at 1 h after the second divided dose of ethanol on GD 57. CPEE resulted in increased liver weight in adult offspring, but produced no difference in fasting blood glucose concentration compared with nutritional control. In the liver, CPEE decreased mRNA expression of IGF-1, IGF-1R, and IGF-2, and increased IRS-2 mRNA expression in male offspring only compared with nutritional control. Female CPEE offspring had decreased INSR hepatic mRNA expression compared with male CPEE offspring. In the prefrontal cortex, IRS-2 mRNA expression was increased in CPEE offspring compared with nutritional control. The data demonstrate that CPEE alters both central and peripheral expression of insulin and IGF signaling molecules at the mRNA level, which may be related to metabolic dysregulation in adult offspring. Furthermore, altered insulin and IGF signaling may be a mechanism of ethanol neurobehavioral teratogenicity.

Differential effects of chronic ethanol exposure on cytochrome P450 2E1 and the hypothalamic–pituitary–adrenal axis in the maternal–fetal unit of the guinea pig

BackgroundEthanol neurobehavioural teratogenicity is a leading cause of developmental mental deficiency, in which the hippocampus is a target site of injury. The multi-faceted mechanism of ethanol teratogenicity is not completely understood. This study tested the hypothesis that chronic ethanol exposure (CEE), via chronic maternal ethanol administration, increases cytochrome P450 2E1 (CYP2E1) expression and alters hypothalamic–pituitary–adrenal (HPA) axis activity in the maternal–fetal unit during the third-trimester-equivalent of gestation. MethodsPregnant Dunkin–Hartley-strain guinea pigs received daily oral administration of ethanol (4g ethanol/kg maternal body weight) or isocaloric-sucrose/pair-feeding (control) throughout gestation (term, about gestational day (GD) 68). On GD 45, 55 and 65, pregnant animals were euthanized 2h after the last daily dose. Maternal and fetal body weights and fetal hippocampal brain weight were determined. Maternal and fetal samples were collected for the determination of liver CYP2E1 enzymatic activity and plasma free cortisol and ACTH concentrations. ResultsCEE, with maternal blood ethanol concentration of 108–124mg/dl at 2h after the last dose, decreased fetal hippocampal weight only at GD 65 and had no effect on fetal body weight compared with control. CYP2E1 activity increased with gestational age in the fetal liver microsomal and mitochondrial fractions. CEE increased CYP2E1 activity in the microsomal and mitochondrial fractions of maternal liver at the three gestational ages and in both hepatic subcellular fractions of the GD 65 fetus compared with control. There was a gestational-age-dependent increase in maternal and fetal plasma free cortisol concentrations, but no effect of CEE compared with control. Maternal and fetal plasma ACTH concentrations were unaffected by CEE compared with control, and were virtually unchanged during the third-trimester-equivalent that was studied. ConclusionThese data demonstrate that, in the pregnant guinea pig, this CEE regimen increases liver CYP2E1 activity, without affecting HPA axis function, in the maternal–fetal unit during near-term gestation. The CEE-induced increase in liver CYP2E1 activity and potential oxidative stress in the maternal–fetal unit may play a role in the pathogenesis of ethanol teratogenicity.

Injurious effects of acute ethanol exposure during late gestation on developing white matter in fetal sheep

Fetal exposure to maternal alcohol intake can be harmful to the developing brain but the effects of acute exposures are less well documented. Our objective was to determine the effects of acute alcohol exposure on developing white matter and to investigate the potential role of pro-inflammatory cytokines. Fifteen pregnant ewes underwent surgery at 110.0±1.0 days of the 147 day gestation for fetal catheterization. Ethanol (1g/kg maternal weight) was administered intravenously to 8 ewes for 1h on 3 consecutive days at 116.0±1.0 days of gestation (0.8 of full term); 7 pregnant control ewes received saline. Fetal brains were collected at necropsy 5 days after the initial ethanol exposure and processed for structural analysis. Maternal and fetal blood ethanol concentrations reached maximal values (0.11±0.01g/dL) 1h after infusions commenced, declining to zero thereafter. Ethanol exposure did not cause fetal hypoxemia, acidemia, hypercapnia, hypoglycemia or hypotension. Subcortical white matter injury, defined as microglia/macrophage infiltration, axonal disruption, increased apoptosis, astrogliosis and altered glial cell morphology, was observed in 4 of the 8 ethanol-exposed fetuses. The injury occupied 6.6–18.3% of the cross-sectional area of cerebral white matter examined and was substantial in 2/8 and modest in 2/8 ethanol-exposed fetuses. Three remaining fetuses exhibited astrogliosis and elevated levels of apoptosis in cerebral white matter. There was a positive correlation between maternal and fetal blood ethanol concentrations and the extent of brain damage. There was no significant elevation in concentrations of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1β and interleukin-6 in fetal plasma. Developing white matter in the late gestation fetus is vulnerable to acute alcohol exposure, but mechanisms remain unclear.

Dose-dependent effects of prenatal ethanol exposure on synaptic plasticity and learning in mature offspring.

We have observed profound deficits in hippocampal synaptic plasticity and one-trial learning in offspring whose mothers drank moderate quantities of ethanol during pregnancy. In the present study, we examined the question of whether lower maternal blood ethanol concentrations (BECs) could produce functional deficits in offspring. Rat dams consumed either a 2%, 3%, or 5% ethanol liquid diet throughout gestation. Three other groups of dams were pair-fed a 0% ethanol liquid diet, and a seventh group consumed lab chow ad libitum. Adult offspring from each diet group were assigned either to studies of evoked [3H]-D-aspartate (D-ASP) release from hippocampal slices or spatial learning studies using the Morris Water Task. Consumption of the 2%, 3%, and 5% ethanol liquid diets produced mean peak maternal BECs of 7, 30 and 83 mg/dL, respectively. Consumption of these ethanol diets had no effect on offspring birthweight, litter size or neonatal mortality. Likewise, evoked D-ASP release from hippocampal slices and performance on a standard version of the Morris Water Task were not affected by prenatal ethanol exposure. By contrast, activity-dependent potentiation of evoked D-ASP release from slices and one-trial learning on a "moving platform" version of the Morris Water Task were markedly reduced in offspring whose mothers consumed the 5% ethanol liquid diet. Intermediate deficits in these two parameters were observed in offspring from the 3% ethanol diet group, whereas offspring from the 2% ethanol diet group were not statistically different than controls. We conclude that the threshold for eliciting subtle, yet significant learning deficits in offspring prenatally exposed to ethanol is less than 30 mg/dL. This BEC is roughly equivalent to drinking 1 to 1.5 ounces of ethanol per day.

Spatial acquisition in the Morris water maze and hippocampal long-term potentiation in the adult guinea pig following brain growth spurt–prenatal ethanol exposure

Previous work has demonstrated that in the guinea pig, chronic prenatal ethanol exposure throughout gestation can result in deficits in spatial learning in the Morris water maze and impaired hippocampal long-term potentiation (LTP). The behavioural effects are known to be dose dependent because water maze deficits occur at a dose of 4 g ethanol/kg maternal body weight/day, but not at a dose of 3 g/kg/day, administered throughout gestation. It is possible that the gradual, progressive development of tolerance to ethanol throughout gestation limits ethanol toxicity, especially for lower doses of ethanol. The present study examined whether neurobehavioural deficits are produced by prenatal ethanol exposure at a dose of 3 g/kg/day, administered only during the brain growth spurt (BGS), a regimen designed to limit the development of ethanol tolerance. Pregnant guinea pigs [term, about gestational day (GD) 68] received oral administration of ethanol (1.5 g/kg maternal body weight/day on GD 43 and 44 and then 3 g/kg maternal body weight/day from GD 45 to 62), isocaloric-sucrose/pair-feeding, or water. Offsprings were studied between postnatal days (PD) 40 and 80. The maternal blood ethanol concentration (BEC) on GD 57 or 58, at 1 h after the daily dose, was 245±19 mg/dl ( n=7). This BGS–prenatal ethanol exposure regimen did not affect spatial learning performance in the Morris water maze over a 7-day test period or in the LTP recorded in the CA1 region of the hippocampus. Thus, even when limiting the development of ethanol tolerance seen with chronic ethanol treatment throughout gestation, ethanol exposure during the BGS does not result in deficits in the behavioural and electrophysiological measures of hippocampal integrity assessed in the present study. These data indicate that in the guinea pig, the BGS may not constitute a critical period of vulnerability for ethanol-induced deficits in spatial learning or hippocampal synaptic plasticity in young adult offspring.

Elimination kinetics of ethanol in pregnant women

To evaluate the pharmacokinetics of ethanol in the early second trimester of pregnancy, ethanol concentrations simultaneously measured in the maternal blood (EtOH-MB) and in the amniotic fluid (EtOH-AF) of six pregnant women were obtained from a previous study in which a single ethanol dose of 300 mg kg −1 body weight was administered orally. For maternal blood ethanol concentration, the kinetic equation was: dC EtOH- MB dt =− k 12+ V max EtOH K m EtOH +C EtOH- MB ×C EtOH- MB +(k 21×C EtOH- AF ) where k 12 and k 21 are, respectively, the rate constant of ethanol transfer from either the central compartment to the peripheral compartment or vice versa; V max EtOH is the maximal velocity for ethanol oxidation; and K m EtOH is the concentration at which half of the maximal rate of ethanol elimination is reached. The maximum concentration of EtOH in AF was 60% lower than in MB ( P=0.036). However, the AUC 0–3.5 h in AF was only 16% lower than the value for MB ( P=0.059). The k 12 (0.20±0.26 h −1) was almost twice faster than k 21 (0.13±0.20 h −1). The V max EtOH was 237.6±71.5 μg ml −1 h −1 and K m EtOH was 3.7±4.7 μg ml −1. Our results imply that in the early second trimester, ethanol metabolism is fast. However, ethanol clearance from the AF is slower than ethanol clearance in MB. This process is widely variable, and our findings may partially explain the wide variability of ethanol’s toxic effects on the fetus.

Chronic Prenatal Ethanol Exposure Alters Ionotropic Glutamate Receptor Subunit Protein Levels in the Adult Guinea Pig Cerebral Cortex

Background: The superfamily of glutamate‐gated ion channels mediates fast excitatory synaptic transmission in the central nervous system and is composed of the NMDA, AMPA, and kainate receptors. Binding studies have shown that chronic prenatal and/or neonatal ethanol exposure produces persistent effects on the numbers of some of these channels. However, whether or not this chronic ethanol exposure produces long‐lasting effects on the expression of specific ionotropic receptor subunits remains an open question.Methods: Timed pregnant Dunkin‐Hartley strain guinea pigs received oral administration of one of the following regimens between gestational days 2 and 67: (1) 4 g of ethanol per kilogram of maternal body weight per day with ad libitum access to pellet food and water (ethanol group), (2) isocaloric sucrose‐ and pair‐feeding with ad libitum access to water (sucrose group), or (3) isovolumetric water with ad libitum access to food and water (water group). The maternal blood ethanol concentration produced by the ethanol regimen was 71 ± 12 mM. Adult offspring were killed on postnatal day 61, and cerebral cortical tissue was analyzed for ionotropic glutamate receptor subunit expression by Western immunoblotting.Results: There was a statistically significant decrease in NR2B subunit protein expression and an increase in GluR2/3 subunit protein expression in the ethanol group. Expression of NR1, NR2A, NR2C, GluR1, GluR6/7, and KA2 subunit proteins was not affected.Conclusions: These results demonstrate that chronic prenatal ethanol exposure produces long‐lasting effects on the subunit composition of NMDA and AMPA receptors in the cerebral cortex of the adult guinea pig.

Brain growth spurt-prenatal ethanol exposure and the guinea pig hippocampal glutamate signaling system

This study tested the hypothesis that prenatal ethanol exposure (PEE) during the brain growth spurt (BGS) in the guinea pig suppresses the glutamate–NMDA receptor–nitric oxide synthase (NOS) signaling system in the developing hippocampus. Pregnant guinea pigs [term, about gestational day (GD) 68] received daily oral administration of 2 g ethanol/kg maternal body weight/day on GD 43 and/or GD 44 and then 4 g ethanol/kg maternal body weight/day from GD 45 to GD 62, isocaloric-sucrose/pair-feeding or water. Offspring were studied at GD 63 (near-term fetus) and postnatal day (PD) 10 (young postnatal life). Maternal blood ethanol concentration during ethanol treatment, pregnancy outcome variables, no change in spontaneous locomotor activity, and decreased brain and cerebral cortical weight data were reported previously [Neurotoxicol. Teratol. 23 (2001) 355]. This BGS-PEE regimen did not affect hippocampal stimulated glutamate release in young postnatal offspring, NMDA receptors as assessed by [ 3H]MK-801 binding, or NOS activity in near-term fetal offspring. Furthermore, BGS-PEE did not affect the number of hippocampal CA1 and CA3 pyramidal cells and dentate gyrus granule cells in defined locations of these three regions in the hippocampal formation. These findings are in contrast to the effects of chronic prenatal exposure to this ethanol regimen throughout gestation, including suppression of the hippocampal glutamate–NMDA receptor–NOS signaling system, decreased number of hippocampal CA1 pyramidal cells, increased spontaneous locomotor activity, and impaired performance in the Morris water maze.

Effects of chronic prenatal ethanol exposure on NMDA receptor number and affinity for [3H]MK-801 in the cerebral cortex of the young postnatal and adult guinea-pig.

The objective of this study was to test the hypothesis that chronic prenatal ethanol exposure (CPEE) produces changes in the number and/or affinity of N-methyl-D-aspartate (NMDA) receptors in the cerebral cortex that are developmental-age-dependent. Timed, pregnant Dunkin-Hartley-strain guinea-pigs received oral intubation of one of the following regimens, given daily as two equally divided doses 2 h apart, from gestational day (GD) 2 to GD 67 (term, ~GD 68): (i) 4 g ethanol kg(-1) maternal bodyweight; (ii) isocaloric sucrose with pair feeding; or (iii) water. Maternal blood ethanol concentration was measured on GD 57 or 58 at 1 h after the daily dose, and was 51.1 +/- 8.5 mM (235 +/- 39 mg dL(-1); n = 8). At postnatal day (PD) 11 (pre-weaning) and PD 61 (adulthood), body, brain and cerebral cortical weights of the offspring were measured. The number of NMDA receptors and their affinity for [(3)H]MK-801 were measured in a crude cerebral cortical membrane preparation using saturation isotherm analysis to determine the B(max) and K(D). Chronic prenatal ethanol exposure decreased offspring brain and cerebral cortical weights at PD 11 and PD 61. At PD 11, there was no CPEE-induced change of [(3)H]MK-801 binding characteristics in the cerebral cortex. At PD 61, both B(max) and K(D) for [(3)H]MK-801 binding to cerebral cortical NMDA receptors were decreased by CPEE compared with the isocaloric sucrose/pair-fed and water treatment groups. Loss of cerebral cortical NMDA receptors and increased affinity of the remaining receptors for [(3)H]MK-801 in the adult guinea-pig, compared with no change in the number or affinity of these receptors in the young postnatal offspring, demonstrated that the effects of CPEE on these ionotropic glutamate receptors are developmental-age-dependent.

Dose‐Dependent Effects of Prenatal Ethanol Exposure on Synaptic Plasticity and Learning in Mature Offspring

Background We have observed profound deficits in hippocampal synaptic plasticity and one‐trial learning in offspring whose mothers drank moderate quantities of ethanol during pregnancy. In the present study, we examined the question of whether lower maternal blood ethanol concentrations (BECs) could produce functional deficits in offspring.Methods Rat dams consumed either a 2%, 3%, or 5% ethanol liquid diet throughout gestation. Three other groups of dams were pair‐fed a 0% ethanol liquid diet, and a seventh group consumed lab chow ad libitum. Adult offspring from each diet group were assigned either to studies of evoked [3H]‐D‐aspartate (D‐ASP) release from hippocampal slices or spatial learning studies using the Morris Water Task.Results Consumption of the 2%, 3%, and 5% ethanol liquid diets produced mean peak maternal BECs of 7, 30 and 83 mg/dL, respectively. Consumption of these ethanol diets had no effect on offspring birthweight, litter size or neonatal mortality. Likewise, evoked D‐ASP release from hippocampal slices and performance on a standard version of the Morris Water Task were not affected by prenatal ethanol exposure. By contrast, activity‐dependent potentiation of evoked D‐ASP release from slices and one‐trial learning on a “moving platform” version of the Morris Water Task were markedly reduced in offspring whose mothers consumed the 5% ethanol liquid diet. Intermediate deficits in these two parameters were observed in offspring from the 3% ethanol diet group, whereas offspring from the 2% ethanol diet group were not statistically different than controls.Conclusions We conclude that the threshold for eliciting subtle, yet significant learning deficits in offspring prenatally exposed to ethanol is less than 30 mg/dL. This BEC is roughly equivalent to drinking 1 to 1.5 ounces of ethanol per day.

Effect of prenatal ethanol exposure during the brain growth spurt of the guinea pig

This study tested the hypothesis that prenatal ethanol exposure during the last third of gestation, including the brain growth spurt (BGS), in the guinea pig produces neurobehavioural teratogenicity, manifesting as brain growth restriction and hyperactivity. Pregnant guinea pigs (term, about gestational day (GD) 68) received oral administration of ethanol (2 g/kg maternal body weight per day on GD 43 and/or GD 44 and then 4 g/kg maternal body weight per day from GD 45 to GD 62), isocaloric-sucrose/pair-feeding, or water. Maternal blood ethanol concentration (BEC) on GD 57 or 58, at 1 h after the daily dose, was 340±76 mg/dl ( n=8). Ethanol treatment decreased brain, cerebral cortical, hippocampal, and cerebellar weights at GD 63 ( P<0.05), and decreased brain and cerebral cortical weights at postnatal day 10 ( P<0.05), with no effect on body weight and no apparent effect on spontaneous locomotor activity. The data demonstrate that, in the guinea pig, prenatal ethanol exposure during the last third of gestation, including the BGS, decreases brain weight that persists into postnatal life, which is associated with growth restriction of the cerebral cortex. However, this prenatal ethanol exposure regimen, including the BGS, does not increase spontaneous locomotor activity in contrast to the persistent hyperactivity that occurs after chronic ethanol exposure throughout gestation.

Chronic Prenatal Ethanol Exposure Increases GABAAReceptor Subunit Protein Expression in the Adult Guinea Pig Cerebral Cortex

Excessive consumption of ethanol during pregnancy can produce teratogenic effects in offspring and is the leading cause of mental deficiency in the Western world. The objective of this study was to examine the effects of chronic prenatal ethanol exposure on the number of GABA(A) receptors and relative protein levels for GABA(A) receptor alpha1 and beta2/3 subunits in the adult guinea pig cerebral cortex. Timed pregnant Dunkin-Hartley strain guinea pigs were given one of the following oral treatments daily throughout gestation: 4 gm of ethanol per kilogram of maternal body weight, isocaloric-sucrose with pair feeding, or isovolumetric water with ad libitum access to food. The ethanol treatment resulted in a peak maternal blood ethanol concentration of 328 +/- 55 mg/dl (71.3 +/- 12.0 mm) on gestational day 57 (term, approximately 68 d). Chronic prenatal exposure to ethanol resulted in increased spontaneous locomotor activity throughout development and decreased cerebral cortical weight in adult offspring. The number of cerebral cortical [(3)H]muscimol binding sites was increased in adult offspring from the ethanol treatment group, and there was a corresponding increase in the amount of GABA(A) receptor alpha1 and beta2/3 subunit proteins in these same animals. For individual offspring, there were correlations between locomotor activity and cerebral cortical weight, as well as between cerebral cortical weight and GABA(A) receptor neurochemistry. There was no effect of chronic prenatal ethanol exposure on [(3)H]MK-801 binding in this tissue. These data demonstrate that chronic prenatal ethanol exposure has long-term consequences on the regulation of GABA(A) receptor expression in the cerebral cortex.

Prenatal exposure to moderate levels of ethanol can have long-lasting effects on learning and memory in adult offspring

Prenatal alcohol exposure can produce long-lasting cognitive and learning impairments in children. Previous studies have suggested that prenatal ethanol exposure can produce neurochemical abnormalities in hippocampal formation. We examined whether performance in the Morris water task, known to be sensitive to interference with hippocampal function, would be affected in adult offspring of rat dams who consumed moderate levels of ethanol through pregnancy. Rat dams consumed one of three diets throughout gestation: (1) Bio-Serve liquid diet containing 5% ethanol (v/v; 26% EDC), which produces a maternal peak blood ethanol concentration of 100 mg/dl; (2) pair fed an isocalorically equivalent amount of 0% ethanol liquid diet; or (3) laboratory chow ad libitum. Adult offspring from each of the three maternal diet conditions were trained in one of three versions of the Morris water task. We employed the “standard” fixed hidden platform task, the moving platform task, and a task that measures competition between cue and place navigation. All three groups learned to navigate normally in the fixed hidden platform version and showed clear preferences for the goal location during no-platform probe trials. In contrast, in the moving platform version and the cue versus place competition task, the prenatally exposed rats were abnormal relative to the rats of the other two maternal diet conditions. In both of these tasks, they behaved in a manner consistent with the idea that acquisition of new place information does not proceed as effectively as in normal rats. These data are consistent with the hypothesis that hippocampal circuitry and neurochemistry related to synaptic plasticity are important targets underlying at least some of the long-lasting deleterious cognitive effects of prenatal alcohol exposure.

Chronic Ethanol Exposure Alters MK-801 Binding Sites in the Cerebral Cortex of the Near-Term Fetal Guinea Pig

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 ± 4 mM (304 ± 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.

Prenatal exposure to moderate levels of ethanol can have long-lasting effects on hippocampal synaptic plasticity in adult offspring.

Prenatal ethanol exposure has been associated with long-lasting intellectual impairments in children. Previous studies using animal models of fetal ethanol exposure suggest that these deficits are, at least in part, linked to neurochemical abnormalities in the hippocampal formation. We explored whether prenatal exposure to moderate quantities of ethanol produced functional deficits at the entorhinal cortical perforant path-dentate granule cell connection by examining some electrophysiological properties, including the induction of long-term potentiation (LTP). Rat dams consumed one of three diets throughout gestation: 1) a BioServ liquid diet containing 5% (v/v) ethanol (26% ethanol-derived calories), which produces a maternal peak blood ethanol concentration of 83 mg/dl; 2) pair-fed an isocalorically equivalent amount of 0% ethanol liquid diet; or 3) Purina rat chow ad libitum. Adult offspring (120-150 days of age) from each experimental diet group were anesthetized with urethane and field excitatory postsynaptic potentials (EPSPs) and population spikes were measured in the dentate gyrus in response to ipsilateral perforant path stimulation. We examined input-output functions using a wide range of single pulse stimulation intensities and induction of LTP using high-frequency stimulation. In the 50-500 microA range of single pulse intensities, there were no significant differences among the diet groups in dentate gyrus evoked potentials. In response to high-frequency stimulation, prenatal ethanol-exposed rats showed a smaller increase in field EPSPs and population spikes compared with rats from either of the two control groups. Thus, prenatal exposure to moderate ethanol levels can produce a long-lasting deficit in synaptic enhancement in a neural pathway believed to be critical in certain forms of learning and memory. This deficit in hippocampal synaptic plasticity may, in part, account for cognitive impairments seen in children whose mothers consumed ethanol during pregnancy.

Prenatal Ethanol Exposure Diminishes Activity‐Dependent Potentiation of Amino Acid Neurotransmitter Release in Adult Rat Offspring

Prenatal ethanol exposure has been associated with long-lasting intellectual impairments in children. Previous studies suggest that these deficits are, in part, linked to neurochemical abnormalities that reduce the ability to sustain long-term potentiation (LTP) in hippocampal formation of adult offspring. One presynaptic component of LTP that manifests during the first half-hour after tetanic stimulation is an enhancement of amino acid neurotransmitter release. Given that the onset of enhanced neurotransmitter release correlates temporally with the decay of hippocampal LTP in prenatal ethanol-exposed offspring, we tested the hypothesis that prenatal ethanol exposure reduces tetanus stimulus-induced potentiation of electrically evoked amino acid release in hippocampal slices. Rat dams consumed 1 of 3 diets throughout gestation: (1) a BioServ liquid diet containing 5% (v/v) ethanol (26% ethanol-derived calories) that produces a maternal peak blood ethanol concentration of 83 mg/dl; (2) pair-fed an isocalorically equivalent amount of 0% ethanol liquid diet; or (3) Purina rat chow ad libitum. Hippocampal slices were prepared from adult offspring from each experimental diet group. Neither the amount of hippocampal slice tissue protein nor the incorporation of [3H]-D-aspartate (D-ASP) was affected by prenatal ethanol exposure. Furthermore, spontaneous efflux and electrically evoked D-ASP release were similar among the three diet groups. However, tetanus stimulus-induced potentiation of evoked D-ASP release in prenatal ethanol-exposed offspring was reduced to about one-third of the potentiation of D-ASP release observed in the control diet groups. These results suggest that prenatal ethanol exposure produces long-lasting deficits in the neurochemical mechanisms responsible for activity-dependent potentiation of amino acid transmitter release without affecting the synaptic machinery responsible for amino acid uptake, storage, and release.

Prenatal ethanol exposure decreases GAP-43 phosphorylation and protein kinase C activity in the hippocampus of adult rat offspring.

Consumption of moderate quantities of ethanol during pregnancy produces deficits in long-term potentiation in the hippocampal formation of adult offspring. Protein kinase C (PKC)-mediated phosphorylation of the presynaptic protein GAP-43 is critical for the induction of long-term potentiation. We tested the hypothesis that this system is affected in fetal alcohol-exposed (FAE) rats by measuring GAP-43 phosphorylation and PKC activity in the hippocampus of adult offspring of rat dams that had consumed one of three diets throughout gestation: (a) a 5% ethanol liquid diet, which produced a maternal blood ethanol concentration of 83 mg/dl (FAE); (b) an isocalorically equivalent 0% ethanol diet (pair-fed); or (c) lab chow ad libitum. Western blot analysis using specific antibodies to PKC-phosphorylated GAP-43 revealed that FAE rats had an approximately 50% reduction in the proportion of phosphorylated GAP-43. Similarly, we found that PKC-mediated incorporation of 32P into GAP-43 was reduced by 85% in hippocampal slices from FAE rats compared with both control groups. FAE animals also showed a 50% reduction in total hippocampal PKC activity, whereas the levels of six major PKC isozymes did not change in any of the diet groups. These results suggest that GAP-43 phosphorylation deficits in rats prenatally exposed to moderate levels of ethanol are not due to alterations in the expression of either the enzyme or substrate protein, but rather to a defect in kinase activation.

Hippocampal nitric oxide synthase in the fetal guinea pig: effects of chronic prenatal ethanol exposure

The effects of chronic maternal administration of ethanol on nitric oxide synthase (NOS) activity and the numbers of NOS containing neurons, and CA1 and CA3 pyramidal neurons in the hippocampus of the near term fetal guinea pig at gestational day (GD) 62 were investigated. Pregnant guinea pigs received oral administration of 4 g ethanol/kg maternal body weight ( n=5), isocaloric sucrose/pair feeding ( n=5) or water ( n=5), or no treatment (NT; n=5) from GD 2 to GD 61. NOS activity in the 25,000× g supernatant of hippocampal homogenate was determined using a radiometric assay. The numbers of NOS containing neurons, and CA1 and CA3 pyramidal neurons were determined using NADPH diaphorase histochemistry and cresyl violet staining, respectively. The chronic ethanol regimen produced a maternal blood ethanol concentration of 193±13 mg/dl at 1 h after the second divided dose on GD 57. Chronic ethanol exposure produced fetal body, brain, and hippocampal growth restriction and decreased fetal hippocampal NOS activity compared with the isocaloric sucrose/pair feeding, water, and NT experimental groups, but did not affect the number of NOS containing and CA1 or CA3 pyramidal neurons. These data demonstrate that, in the near term fetus, chronic maternal administration of ethanol suppresses hippocampal NOS activity and consequent formation of NO, without loss of NOS containing neurons and prior to loss of CA1 pyramidal neurons that occurs in the adult.

Effect of chronic maternal ethanol administration on glutamate and N-methyl- d-aspartate binding sites in the hippocampus of the near-term fetal guinea pig

The objective of this study was to determine the effect of chronic maternal administration of ethanol on hippocampal l-glutamate (glutamate) and N-methyl- d-aspartate (NMDA) binding sites in the near-term fetal guinea pig. Starting on gestational day (GD) 2, pregnant guinea pigs received one of the following oral treatments up to and including GD 62 (term, about GD 68): 4 g ethanol · kg maternal body weight −1 · day −1; isocaloric sucrose and pair-feeding; or water. Maternal blood ethanol concentration was determined at 1 h after the daily ethanol dose on GD 59. Fetuses were studied at GD 63 (mature, near-term fetus). Fetal body weight and brain weight were determined. The density ( B max) and affinity ( K d) of the glutamate and NMDA binding sites in the fetal hippocampus were measured using a radioligand membrane binding assay; saturation analysis was conducted on hippocampal synaptic membrane preparation (HSMP). Maternal blood ethanol concentration on GD 59 was 269 ± 111 (SD) mg/dl (59 ± 24 mM). There was no maternal or embryonic fetal lethality in any of the three treatment groups, and ethanol treatment did not affect maternal body weight gain compared with sucrose or water treatment. Fetal brain weight, but not body weight, was decreased in the ethanol treatment group compared with the sucrose and water treatment groups. The B max values of the glutamate and NMDA binding sites were decreased in the ethanol treatment group compared with the sucrose and water treatment groups; there was no difference in the K d values of the glutamate and NMDA binding sites among the three treatment groups. The data demonstrate that, in the guinea pig, chronic maternal administration of a binge-type ethanol regimen throughout gestation restricts brain growth and decreases the density of glutamate and NMDA binding sites in the hippocampus, as manifested in the mature fetus.