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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
Alcohol, Vol. 12, No. 4, pp. 377-382, 1995 Copyright • 1995ElsevierScienceLtd Printed in the USA. All rights reserved 0741-8329/95 $9.50 + .00
Pergamon 0741-8329(95)00021-6
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 SHIRIN
ABDOLLAH
AND
JAMES
F. B R I E N 1
Department o f Pharmacology and Toxicology, Faculty o f Medicine, Queen's University, Kingston, Ontario, Canada K7L 3N6 R e c e i v e d 18 O c t o b e r 1994; A c c e p t e d 26 J a n u a r y 1995 ABDOLLAH, S. AND J. F. BRIEN. Effect of chronic maternal ethanol administration on glutamate and N-methyi-Daspartate binding sit~ in the hippocampus of the near-term fetal guinea pig. ALCOHOL 12(4) 377-382, 1995.-The objective of this study was to determine the effect of chronic maternal admini.~ration of ethanol on hippoeampal 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 -t • day-~; isocaloric sucrose and pair-feeding; or water. Maternal blood ethanol concentration was determined at I 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 (BAR)and affinity (Ko) of the glutamate and NMDA binding sites in the fetal hippocampus were measured using a radioligand membrane binding assay; saturation ~nalysis was conducted on hippocampal synal~c 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 m 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 Kd 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. Glutamate N-Met hyl-D-aspartate Chronic ethanol exposure
Binding sites
Hippocampus
Guinea pig
Near-term fetus
ing characteristic o f ethanol teratogenesis. One o f the target sites o f ethanol CNS teratogenesis is the hippocampus (27), which is a cortical structure that is important in the processes o f learning, memory, and control o f behaviour (4,25). Prenatal ethanol exposure results in several structural and functional changes in the hippocampus that have been observed in
E T H A N O L teratogenesis can manifest as the fetal alcohol syndrome in the h u m a n (15), consisting principally o f central nervous system (CNS) dysfunction, pre- and postnatal growth deficiency, and c r a n i o f a d a l dysmorphology (8). The C N S dysfunction, which can manifest as mental deficiency, hyperactivity, and m o t o r incoordination, is probably the most debilitat-
To whom requests for reprints should be addressed. 377
378
ABDOLLAH AND BRIEN
adult rat or guinea pig viz: morphological damage involving CAI pyramidal cell loss (1,5), decreased dendritic spines in the CA1 region (3), and abnormal mossy fibre formation (26); and neurochemical and electrophysiological alterations including decreased number of NMDA-sensitive [3H]giutamate binding sites (21) and decreased long-term potentiation (LTP) (23), a form of synaptic plasticity that appears to play an important role in memory formation and learning. Injury to the developing hippocampus may be one of the underlying causes of the learning, memory, and behavioural dysfunction of ethanol CNS teratogenesis (12). The mechanism by which prenatal ethanol exposure produces its structural and functional changes in the hippocampus may involve the glutamatergic neuronal pathway (18). L-Glutamate (glutamate) is the primary excitatory neurotransmitter in the hippocampus (9). Glutamate acts in the CNS by binding to and activating excitatory amino acid receptors, which can be broadly classified as N-methyl-D-aspartate (NMDA) and non-NMDA types of receptors (9). NMDA receptors appear to be involved in mediating Glu's action in the formation of LTP and in brain development (10,17). Optimal activation of the glutamate neurotransmitter-NMDA receptor system is considered to be important for normal brain development, such that underactivation or overactivation of this system during development may lead to abnormalities in CNS structure and function (17). Our laboratory is focused on testing the hypothesis that ethanol neurobehavioural teratogenesis involving the hippocampus is related to a decrease in hippocampal glutamate and NMDA binding sites. The objective of the current study was to determine the effect of chronic administration of ethanol to pregnant guinea pigs on the glutamate and NMDA binding sites in the hippocampus of the mature, near-term fetus. The guinea pig was selected as the experimental animal due to its extensive prenatal brain development including the brain growth spurt, which peaks around gestational day 45-53 (11). Furthermore, we have shown that chronic administration of 4 g ethanol • kg maternal body weight-l . day-m to the pregnant guinea pig produces the following: no maternal or embryonic fetal lethality, hyperactivity in the offspring that persists into adulthood, and structural injury in the hippocampus manifesting as a 25% decrease in the number of pyramidal neurons in the CAI region (1,7). An optimized radioligand membrane binding assay was used in this study, involving [3H]giutamate and a hippocampal synaptic membrane preparation (HSMP) of the near-term fetal guinea pig. METHOD
Chemicals and Solutions The chemicals and solutions used in this study are the same as those described in our previous article (2).
Experimental Animals Nulliparous female Dunkin-Hartley strain guinea pigs (450-600 g body weight) were bred with male Dunkin-Hartley strain guinea pigs using an established procedure (13). Gestational day (GD) 0 was taken as the last day of full vaginal membrane opening. On GD 1, pregnant animals were housed singly in stainless steel wire cages with a 12L : 12D cycle with lights on at 0700 h. All animals were cared for according to the principles and guidelines of the Canadian Council on Animal Care, and the experimental protocol was approved by the Queen's University Animal Care Committee.
Animal Treatment Regimens On GD 2, each animal was assigned to receive one of the following dally treatment regimens, via intubation into the oral cavity, up to and including GD 62 (term, about GD 68). For the ethanol regimen, 4 g ethanol • kg maternal body weight-m . day-i was administered as two equally divided doses given 2 h apart with ad lib access to pellet food (Purina Guinea Pig Chow 5025 ®) and water. For the isocaloric sucrose and pair-feeding regimen, the animal was paired to an ethanol-treated guinea pig, received sucrose as two divided doses given 2 h apart that was isocaloric and isovolumetric to the dally dose of ethanol, and was given food in an amount equivalent to that consumed dally by the ethanol-treated animal plus free access to water. For the water regimen, water was administered as two divided doses 2 h apart with ad lib access to food and water. The daily treatment commenced between 1100 and 1300 h, and involved intubation of the appropriate solution into the oral cavity with a pediatric feeding tube. The ethanol (30o/o, v/v) and sucrose (42%, w/v) solutions were prepared in tap water. Maternal body weight and membrane status of the pregnant guinea pigs were checked daily. On GD 59, a maternal blood sample was obtained from an ear vein at 1 h after the second divided dose of ethanol. The ethanol concentration in the blood sample was determined in duplicate using an established gas-liquid chromatographic method, in which the recovery, within-day coefficient of variation, and lower limit of quantitative sensitivity were 98.2%, _<7.0%, and 0.005 mg/mi, respectively (22).
Hippocampal Synaptic Membrane Preparation (HSMP) On GD 63, each pregnant guinea pig was killed by decapitation, and a maternal blood sample was obtained from each ethanol-treated animal for the determination of blood ethanol concentration. The gravid uterus was exposed via a midline abdominal incision; each fetus was removed via a uterine incision and weighed; and the gender of each fetus was determined. Each fetus was killed by decapitation, and its brain was excised, weighed, and placed in ice-cold 50 mM Trisacetate buffer (pH 7.4). The hippocampi were dissected, pooled for each litter, weighed, and then used to prepare a synaptic membrane preparation by an established method (28) that was used for the near-term fetal guinea pig as previously described (2).
Radioligand Membrane Binding Assay of Hippocampal Glutamate and NMDA Binding Sites To characterize the glutamate and NMDA binding sites with respect to their density (Bin0 and affinity (Kd), saturation analysis was conducted. HSMP (about 40/~g protein) suspension in 50 mM Tris-acetate buffer (pH 7.4) was incubated with different concentrations of [3H]glutamate (1-200 nM) in a fined volume of 500 ~1 at 4°C for 10 rain. The incubation was terminated by the addition of 3 ml of ice-cold 50 mM Trisacetate, followed by vacuum f'dtration at 38 cmHg through a Whatman GF/B glass fibre f'dter. The f'dter and retained material were rinsed three times with 3-ml aliquots of ice-cold 50 mM Tris-acetate. The radioactivity bound to the HSMP on the washed filter was measured by liquid scintillation spectrometry as previously described (2). The radioligand memhrane binding assay for each [3H]glutamate concentration was conducted in duplicate or triplicate.
EFFECT OF CHRONIC IVlATERNAL ETHANOL ADMINISTRATION
379
Data Analysis
lOO
The maternal blood ethanol concentration data are presented as the mean + SD. Maternal body weight gain is expressed as cumulative change in body weight at successive l-week intervals compared with the body weight at GD 1. The data are presented as group means + SD for the ethanol, sucrose, and water treatment groups, and were statistically analyzed by two-way analysis of variance (ANOVA) with treatment as an independent factor and time as a repeated factor. The average fetal body weight and brain weight for each litter were calculated, and the data are presented as group means ± SD for the ethanol, sucrose, and water treatment groups. For each of fetal body weight and brain weight, the results of the three treatment groups were compared by a randomized-design one*way ANOVA followed by NewmanKeuls test for a significant F statistic (p < 0.05). Specific [3H]glutamate binding and NMDA-sensitive [3H]glutamate binding were calculated as previously described (2), and are expressed as pmol [3H]glutamate bound/mg HSMP protein. The analysis of the saturation data was conducted as previously described (2). For each of the glutamate and NMDA binding sites, the Bm~ and Kd values are expressed as pmol [3H]glutamate hound/mg HSMP protein and nM [3H]glutamate, respectively, and the data are presented as group means + SD for the ethanol, sucrose, and water treatment groups. For each of the glutamate and NMDA binding sites, the B=~ and Kd values were statistically compared among the three treatment groups using a randomized-design one*way ANOVA followed by Newman-Keuls test for a significant F statistic (p _< 0.05). All data were initially tested for homogeneity of
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FIG. 2. Body weight and brain weight for near-term fetuses (GD 63) of pregnant guinea pigs that received oral intubation of 4 g ethanol • kg-i . day-1 (n = 6 litters), isocaloric sucrose/palr-fceding (n = 6 litters), or water (n = 4 litters) from GD 2 to GD 62. Fetal body weight and brain weight are expressed as average fetal body weight and brain weight per litter, respectively. The data are presented as group means + SD. There was a statistical difference (*) in the fetal brain weight for the ethanol treatment group compared with the sucrose and water treatment groups, F(2, 13) = 30.41,p < 0.001.
RESULTS
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FIG. 1. Maternal body weight gain for pregnant guinea pigs that received oral intubation of 4 g ethanol, kg maternal body weight- I • day -~, isocaloric sucrose/pair-f~.=eding,or water from GD 2 to GD 62. Maternal body weight gain is expressed as cumulative change in body weight at successive l-week intervals compared with maternal body weight at GD 1. The data are presented as group means + SD. The maternal body weight values at GD 1 for the ethanol-, sucrose-, and water-treated groups were 520 + 22 g (n = 6), 530 + 41 g (n = 6), and 544 + 71 g (n = 5), respectively. There was a statistically significant effect of time (week of gestation), F(8, 112) = 300.42,p < 0.05.
The regimen of 4 g ethanol • kg maternal body weight- ~ • day -~, administered to the pregnant guinea pig throughout gestation, produced a maternal blood ethanol concentration of 269 ± 111 m g / d l ( 5 8 ± 2 4 m M ) ( n -- 6) at 1 h a f t e r t h e second divided dose, as determined on GD 59. This value is similar to the maternal blood ethanol concentration of 247 mg/dl (54 raM), determined for this ethanol regimen on GD 56 in our previous study of the dose*dependent effects of prenatal ethanol exposure on p r e g n a n ~ outcome and offspring hehaviour (1,7). Fetal hippoeampal tissue was obtained on GD 63, 24 h after the last ethanol dose, and there was no measurable ethanol in maternal blood. The maternal ~unulative body weight change data for the ethanol, sucrose, and water treatments are presented in Fig. 1, and there was no difference in the time course profiles among the three treatments. There was no maternal or ~nbryonic fetal lethality for the three treatment groups. Litter size of the pregnant guinea pigs in the ethanol, sucrose, and water treatment groups was 2.7 ± 1.0 (n = 6), 3.5 ± 1.0 (n ffi 6), and 3.6 ± 1.5 (n -- 5), respectively, and there was no difference among the three treatment groups. In regard to the gender of the fetuses, the total numbers of males and females for all the litters of the ethanol, sucrose, and water treatment groups were 6 and 9 (n = 6 litters); 13 and 8 (n = 6 litters); and 7 and 7 (n = 4 litters), respectively. There was no statistical difference in the percentage of females and percentage of
380
ABDOLLAH AND BRIEN
males per litter among the ethanol, sucrose, and water treatment groups: 51 + 35070females and 49 ± 35070males for the ethanol group; 32 ± 31070 females and 68 ± 31070 males for the sucrose group; 56 ± 43070 females and 44 ± 43070 males for the water group. The fetal body weight and brain weight data at GD 63 for the ethanol, sucrose, and water treatment groups are presented in Fig. 2. There was no difference in fetal body weight among the three treatment groups. There was, however, about a 20070 decrease in fetal brain weight for the ethanol treatment group compared with each of the sucrose and water treatment groups (p < 0.05). [3H]Glutamate bound specifically and in an NMDA-sensitive manner to the HSMP of the guinea pig fetus at GD 63 for each of the ethanol, sucrose, and water treatment groups. The magnitude of specific [3H]glutamate binding and NMDAsensitive [3H]glutamate binding increased with [3H]glutamate concentration, reaching apparent saturation between 100 and 200 nM [3Hlglutamate (Fig. 3). The Bm~ and K d data of each of the glutamate and NMDA binding sites of the fetal guinea pig hippocampus at GD 63 for the three treatment groups are presented in Figs. 4 and 5, respectively. The Bm~ values of the glutamate and NMDA binding sites of the ethanol treatment
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FIG. 4. Brr~ and Kd values for the hippocampal glutamate binding sites of near-term fetuses (GD 63) of pregnant guinea pigs that. received oral intubation of 4 g ethanol • kg-* • day-~ (n = 5 litters), isocaloric sucrose/pair-feeding (n = 5 litters), or water (n = 4 litters) from GD 2 to GD 62. The data are presented as group means + SD. There was a statistical difference (*) in the 8 ~ value for the ethanol treatment group compared with the sucrose and water treatment groups, F(2, ll) = 5.14,p < 0.05.
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DISCUSSION
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group were 39°70 and 31 °70 lower, respectively, compared with the sucrose treatment group (p _< 0.05), and were 39070 and 43% lower, respectively, compared with the water treatment group (p < 0.05). There was no difference in the Kd values of the glutamate and NMDA.binding sites among the ethanol, sucrose, and water treatment groups.
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FIG. 3. Effects o f glutamate concentration on specific [3H]glutamate binding and NMDA-sensitive [3H]glutamate binding (saturation plots) in HSMP o f the near-term fetus (GD 63) o f pregnant guinea pigs that received chronic oral intubation o f 4 g ethanol • kg- i . day- i (n = 1 litter), isocaloric sucrose/pair-feeding (n -- 1 litter), or water (n = 1 litter) from GD 2 to GD 62. Curves o f best fit were obtained by computer-assisted, nonlinear regression analysis o f the data.
The overall goal of our research program is to test the hypothesis that ethanol neurobehavioural teratogenesis involving the hippocampus is due, at least in part, to a decrease in hippocampal glutamatergic function. The objective of this study was to determine the effect of chronic ethanol administration to pregnant guinea pigs on the density and affinity of glutamate and NMDA binding sites in the hippocampus of the mature, near-term fetus. The guinea pig was studied because of its extensive prenatal brain development, including the brain growth spurt. The data of the present study confirm that the selected regimen of 4 g ethanol • kg-~ • day-~ produces CNS teratogenesis without causing maternal or embryonic/ fetal lethality, as previously reported 0,7). This ethanol regimen did not change the size or gender ratio of the litter or fetal body weight. The regimen decreased brain weight com-
EFFECT OF CHRONIC M A T E R N A L E T H A N O L A D M I N I S T R A T I O N GLUTAMATE BINDING SITES 6 ¢
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FIG. 5. Bm~ and Kd values for the hippocampal NMDA binding sites of near-term fetuses (GD 63) of pregnant guinea pigs that received oral intubation of 4 g e t h a n o l • kg-m • day-m (n = 5 litters), isocaloric sucrose/pair-feeding (n = 5 litters), or water (n = 4 fitters) from GD 2 to GD 62. The data are presented as group means + SD. There was a statistical differev:ce (*) in the Bm~ value for the ethanol treatment group compared with the sucrose and water treatment groups, F(2, 11) = 6.57,p < 0.05.
pared with the isocaloric sucrose/pair-feeding and water treatment control groups, which is indicative of microencephaly. Ethanol-induced microencephaly, determined by decreased brain weight, also occurs in the neonatal rat pup following ethanol exposure during the period encompassing the peak of the brain growth spurt (postnatal day 4 to 10), with no change in body weight (6). The selective ethanol-induced microencephaly observed in the mature, near-term fetal guinea pig could be the consequence of exposure of the brain to ethanol during the brain growth spurt, which occurs prenatally in this rodent species (11). The hippocampus was selected for study because it is one of the target sites of ethanol teratogenesis. Most of the studies investigating the neurochemical, electrophysiologlcal, and morphological changes in the hippocampus, produced by prenatal ethanol exposure, have focused on the offspring in post-
381
natal life (14,19,21,24). In the rat, prenatal ethanol exposure decreases NMDA-sensitive [3H]glutamate binding in various regions of the dorsal hippocampal formation by 18% to 21%, as determined in adulthood (21). In view o f the proposed neurotrophic role of the glutamate neurotransmitter-NMDA receptor system during brain development (17), it would appear to be important to elucidate the effect o f prenatal ethanol exposure, via maternal ethanol administration, on the glutamate and NMDA binding sites in the fetal hippocampus. We studied the mature, near-term fetal guinea pig because the density of the glutamate and NMDA binding sites appears to be greater at this prenatal time of development, based on our previous ontogeny study (2). Chronic maternal ethanol administration decreased the density of the glutamate and NMDA binding sites in the fetal hippocampus compared with the control, without affecting the affinity o f these binding sites. These findings are indicative of decreased synthesis a n d / or increased catabolism of excitatory amino acid receptors, and NMDA receptors in particular, without affecting the binding characteristics of the glutamate recognition sites on these receptors. This appears to be the first reported study of the effect of prenatal ethanol exposure on fetal hippocampal glutamate and NMDA binding sites. Our results are in agreement with the reported decrease in the density of dorsal hippocampal NMDA binding sites (21) and whole-brain glutamate binding sites (16) in the adult and neonatal rat, respectively, produced by prenatal exposure to ethanol. Furthermore, in both of these studies, there was no change in the affinity of the binding sites (16,21). The decreased density of the glutamate and NMDA binding sites that we report here, together with the previously reported depressant effect of ethanol on stimulated and basal release of glutamate in the hippocampus of the mature, near-term fetal guinea pig (20), could lead to decreased function of the glutamaterglc system in this brain region, including suppression of the neurotrophic action of the glutamate neurotransmitter-NMDA receptor system. If this latter action occurs, it would be an important contributing factor in the mechanism of ethanol teratogenesis involving the hippocampus. In conclusion, chronic administration of 4 g ethanol • kgday-~ to the pregnant guinea pig throughout gestation produces microencephaly and decreased density of the hippocampal glutamate and NMDA binding sites in the mature, nearterm fetus. Further studies are required to determine whether this decreased density of fetal hippocampal glutamate and NMDA binding sites persists into postnatal fife, and to determine the relationship between the decreased density of hippocampal glutamate and NMDA binding sites and the hyperactivity and hippocampal structural injury, observed in adulthood, that result from chronic prenatal ethanol exposure. ACKNOWLEDGEMENTS
This research was supported by an operating grant from the Medical Research Council of Canada (MT-8073). S.A. was the recipient of an Eldon Boyd Fellowship (Department of Pharmacology and Toxicology) and a Queen's Graduate Award.
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Pergamon 0741-8329(95)00021-6
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 SHIRIN
ABDOLLAH
AND
JAMES
F. B R I E N 1
Department o f Pharmacology and Toxicology, Faculty o f Medicine, Queen's University, Kingston, Ontario, Canada K7L 3N6 R e c e i v e d 18 O c t o b e r 1994; A c c e p t e d 26 J a n u a r y 1995 ABDOLLAH, S. AND J. F. BRIEN. Effect of chronic maternal ethanol administration on glutamate and N-methyi-Daspartate binding sit~ in the hippocampus of the near-term fetal guinea pig. ALCOHOL 12(4) 377-382, 1995.-The objective of this study was to determine the effect of chronic maternal admini.~ration of ethanol on hippoeampal 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 -t • day-~; isocaloric sucrose and pair-feeding; or water. Maternal blood ethanol concentration was determined at I 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 (BAR)and affinity (Ko) of the glutamate and NMDA binding sites in the fetal hippocampus were measured using a radioligand membrane binding assay; saturation ~nalysis was conducted on hippocampal synal~c 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 m 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 Kd 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. Glutamate N-Met hyl-D-aspartate Chronic ethanol exposure
Binding sites
Hippocampus
Guinea pig
Near-term fetus
ing characteristic o f ethanol teratogenesis. One o f the target sites o f ethanol CNS teratogenesis is the hippocampus (27), which is a cortical structure that is important in the processes o f learning, memory, and control o f behaviour (4,25). Prenatal ethanol exposure results in several structural and functional changes in the hippocampus that have been observed in
E T H A N O L teratogenesis can manifest as the fetal alcohol syndrome in the h u m a n (15), consisting principally o f central nervous system (CNS) dysfunction, pre- and postnatal growth deficiency, and c r a n i o f a d a l dysmorphology (8). The C N S dysfunction, which can manifest as mental deficiency, hyperactivity, and m o t o r incoordination, is probably the most debilitat-
To whom requests for reprints should be addressed. 377
378
ABDOLLAH AND BRIEN
adult rat or guinea pig viz: morphological damage involving CAI pyramidal cell loss (1,5), decreased dendritic spines in the CA1 region (3), and abnormal mossy fibre formation (26); and neurochemical and electrophysiological alterations including decreased number of NMDA-sensitive [3H]giutamate binding sites (21) and decreased long-term potentiation (LTP) (23), a form of synaptic plasticity that appears to play an important role in memory formation and learning. Injury to the developing hippocampus may be one of the underlying causes of the learning, memory, and behavioural dysfunction of ethanol CNS teratogenesis (12). The mechanism by which prenatal ethanol exposure produces its structural and functional changes in the hippocampus may involve the glutamatergic neuronal pathway (18). L-Glutamate (glutamate) is the primary excitatory neurotransmitter in the hippocampus (9). Glutamate acts in the CNS by binding to and activating excitatory amino acid receptors, which can be broadly classified as N-methyl-D-aspartate (NMDA) and non-NMDA types of receptors (9). NMDA receptors appear to be involved in mediating Glu's action in the formation of LTP and in brain development (10,17). Optimal activation of the glutamate neurotransmitter-NMDA receptor system is considered to be important for normal brain development, such that underactivation or overactivation of this system during development may lead to abnormalities in CNS structure and function (17). Our laboratory is focused on testing the hypothesis that ethanol neurobehavioural teratogenesis involving the hippocampus is related to a decrease in hippocampal glutamate and NMDA binding sites. The objective of the current study was to determine the effect of chronic administration of ethanol to pregnant guinea pigs on the glutamate and NMDA binding sites in the hippocampus of the mature, near-term fetus. The guinea pig was selected as the experimental animal due to its extensive prenatal brain development including the brain growth spurt, which peaks around gestational day 45-53 (11). Furthermore, we have shown that chronic administration of 4 g ethanol • kg maternal body weight-l . day-m to the pregnant guinea pig produces the following: no maternal or embryonic fetal lethality, hyperactivity in the offspring that persists into adulthood, and structural injury in the hippocampus manifesting as a 25% decrease in the number of pyramidal neurons in the CAI region (1,7). An optimized radioligand membrane binding assay was used in this study, involving [3H]giutamate and a hippocampal synaptic membrane preparation (HSMP) of the near-term fetal guinea pig. METHOD
Chemicals and Solutions The chemicals and solutions used in this study are the same as those described in our previous article (2).
Experimental Animals Nulliparous female Dunkin-Hartley strain guinea pigs (450-600 g body weight) were bred with male Dunkin-Hartley strain guinea pigs using an established procedure (13). Gestational day (GD) 0 was taken as the last day of full vaginal membrane opening. On GD 1, pregnant animals were housed singly in stainless steel wire cages with a 12L : 12D cycle with lights on at 0700 h. All animals were cared for according to the principles and guidelines of the Canadian Council on Animal Care, and the experimental protocol was approved by the Queen's University Animal Care Committee.
Animal Treatment Regimens On GD 2, each animal was assigned to receive one of the following dally treatment regimens, via intubation into the oral cavity, up to and including GD 62 (term, about GD 68). For the ethanol regimen, 4 g ethanol • kg maternal body weight-m . day-i was administered as two equally divided doses given 2 h apart with ad lib access to pellet food (Purina Guinea Pig Chow 5025 ®) and water. For the isocaloric sucrose and pair-feeding regimen, the animal was paired to an ethanol-treated guinea pig, received sucrose as two divided doses given 2 h apart that was isocaloric and isovolumetric to the dally dose of ethanol, and was given food in an amount equivalent to that consumed dally by the ethanol-treated animal plus free access to water. For the water regimen, water was administered as two divided doses 2 h apart with ad lib access to food and water. The daily treatment commenced between 1100 and 1300 h, and involved intubation of the appropriate solution into the oral cavity with a pediatric feeding tube. The ethanol (30o/o, v/v) and sucrose (42%, w/v) solutions were prepared in tap water. Maternal body weight and membrane status of the pregnant guinea pigs were checked daily. On GD 59, a maternal blood sample was obtained from an ear vein at 1 h after the second divided dose of ethanol. The ethanol concentration in the blood sample was determined in duplicate using an established gas-liquid chromatographic method, in which the recovery, within-day coefficient of variation, and lower limit of quantitative sensitivity were 98.2%, _<7.0%, and 0.005 mg/mi, respectively (22).
Hippocampal Synaptic Membrane Preparation (HSMP) On GD 63, each pregnant guinea pig was killed by decapitation, and a maternal blood sample was obtained from each ethanol-treated animal for the determination of blood ethanol concentration. The gravid uterus was exposed via a midline abdominal incision; each fetus was removed via a uterine incision and weighed; and the gender of each fetus was determined. Each fetus was killed by decapitation, and its brain was excised, weighed, and placed in ice-cold 50 mM Trisacetate buffer (pH 7.4). The hippocampi were dissected, pooled for each litter, weighed, and then used to prepare a synaptic membrane preparation by an established method (28) that was used for the near-term fetal guinea pig as previously described (2).
Radioligand Membrane Binding Assay of Hippocampal Glutamate and NMDA Binding Sites To characterize the glutamate and NMDA binding sites with respect to their density (Bin0 and affinity (Kd), saturation analysis was conducted. HSMP (about 40/~g protein) suspension in 50 mM Tris-acetate buffer (pH 7.4) was incubated with different concentrations of [3H]glutamate (1-200 nM) in a fined volume of 500 ~1 at 4°C for 10 rain. The incubation was terminated by the addition of 3 ml of ice-cold 50 mM Trisacetate, followed by vacuum f'dtration at 38 cmHg through a Whatman GF/B glass fibre f'dter. The f'dter and retained material were rinsed three times with 3-ml aliquots of ice-cold 50 mM Tris-acetate. The radioactivity bound to the HSMP on the washed filter was measured by liquid scintillation spectrometry as previously described (2). The radioligand memhrane binding assay for each [3H]glutamate concentration was conducted in duplicate or triplicate.
EFFECT OF CHRONIC IVlATERNAL ETHANOL ADMINISTRATION
379
Data Analysis
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The maternal blood ethanol concentration data are presented as the mean + SD. Maternal body weight gain is expressed as cumulative change in body weight at successive l-week intervals compared with the body weight at GD 1. The data are presented as group means + SD for the ethanol, sucrose, and water treatment groups, and were statistically analyzed by two-way analysis of variance (ANOVA) with treatment as an independent factor and time as a repeated factor. The average fetal body weight and brain weight for each litter were calculated, and the data are presented as group means ± SD for the ethanol, sucrose, and water treatment groups. For each of fetal body weight and brain weight, the results of the three treatment groups were compared by a randomized-design one*way ANOVA followed by NewmanKeuls test for a significant F statistic (p < 0.05). Specific [3H]glutamate binding and NMDA-sensitive [3H]glutamate binding were calculated as previously described (2), and are expressed as pmol [3H]glutamate bound/mg HSMP protein. The analysis of the saturation data was conducted as previously described (2). For each of the glutamate and NMDA binding sites, the Bm~ and Kd values are expressed as pmol [3H]glutamate hound/mg HSMP protein and nM [3H]glutamate, respectively, and the data are presented as group means + SD for the ethanol, sucrose, and water treatment groups. For each of the glutamate and NMDA binding sites, the B=~ and Kd values were statistically compared among the three treatment groups using a randomized-design one*way ANOVA followed by Newman-Keuls test for a significant F statistic (p _< 0.05). All data were initially tested for homogeneity of
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FIG. 2. Body weight and brain weight for near-term fetuses (GD 63) of pregnant guinea pigs that received oral intubation of 4 g ethanol • kg-i . day-1 (n = 6 litters), isocaloric sucrose/palr-fceding (n = 6 litters), or water (n = 4 litters) from GD 2 to GD 62. Fetal body weight and brain weight are expressed as average fetal body weight and brain weight per litter, respectively. The data are presented as group means + SD. There was a statistical difference (*) in the fetal brain weight for the ethanol treatment group compared with the sucrose and water treatment groups, F(2, 13) = 30.41,p < 0.001.
RESULTS
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FIG. 1. Maternal body weight gain for pregnant guinea pigs that received oral intubation of 4 g ethanol, kg maternal body weight- I • day -~, isocaloric sucrose/pair-f~.=eding,or water from GD 2 to GD 62. Maternal body weight gain is expressed as cumulative change in body weight at successive l-week intervals compared with maternal body weight at GD 1. The data are presented as group means + SD. The maternal body weight values at GD 1 for the ethanol-, sucrose-, and water-treated groups were 520 + 22 g (n = 6), 530 + 41 g (n = 6), and 544 + 71 g (n = 5), respectively. There was a statistically significant effect of time (week of gestation), F(8, 112) = 300.42,p < 0.05.
The regimen of 4 g ethanol • kg maternal body weight- ~ • day -~, administered to the pregnant guinea pig throughout gestation, produced a maternal blood ethanol concentration of 269 ± 111 m g / d l ( 5 8 ± 2 4 m M ) ( n -- 6) at 1 h a f t e r t h e second divided dose, as determined on GD 59. This value is similar to the maternal blood ethanol concentration of 247 mg/dl (54 raM), determined for this ethanol regimen on GD 56 in our previous study of the dose*dependent effects of prenatal ethanol exposure on p r e g n a n ~ outcome and offspring hehaviour (1,7). Fetal hippoeampal tissue was obtained on GD 63, 24 h after the last ethanol dose, and there was no measurable ethanol in maternal blood. The maternal ~unulative body weight change data for the ethanol, sucrose, and water treatments are presented in Fig. 1, and there was no difference in the time course profiles among the three treatments. There was no maternal or ~nbryonic fetal lethality for the three treatment groups. Litter size of the pregnant guinea pigs in the ethanol, sucrose, and water treatment groups was 2.7 ± 1.0 (n = 6), 3.5 ± 1.0 (n ffi 6), and 3.6 ± 1.5 (n -- 5), respectively, and there was no difference among the three treatment groups. In regard to the gender of the fetuses, the total numbers of males and females for all the litters of the ethanol, sucrose, and water treatment groups were 6 and 9 (n = 6 litters); 13 and 8 (n = 6 litters); and 7 and 7 (n = 4 litters), respectively. There was no statistical difference in the percentage of females and percentage of
380
ABDOLLAH AND BRIEN
males per litter among the ethanol, sucrose, and water treatment groups: 51 + 35070females and 49 ± 35070males for the ethanol group; 32 ± 31070 females and 68 ± 31070 males for the sucrose group; 56 ± 43070 females and 44 ± 43070 males for the water group. The fetal body weight and brain weight data at GD 63 for the ethanol, sucrose, and water treatment groups are presented in Fig. 2. There was no difference in fetal body weight among the three treatment groups. There was, however, about a 20070 decrease in fetal brain weight for the ethanol treatment group compared with each of the sucrose and water treatment groups (p < 0.05). [3H]Glutamate bound specifically and in an NMDA-sensitive manner to the HSMP of the guinea pig fetus at GD 63 for each of the ethanol, sucrose, and water treatment groups. The magnitude of specific [3H]glutamate binding and NMDAsensitive [3H]glutamate binding increased with [3H]glutamate concentration, reaching apparent saturation between 100 and 200 nM [3Hlglutamate (Fig. 3). The Bm~ and K d data of each of the glutamate and NMDA binding sites of the fetal guinea pig hippocampus at GD 63 for the three treatment groups are presented in Figs. 4 and 5, respectively. The Bm~ values of the glutamate and NMDA binding sites of the ethanol treatment
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FIG. 4. Brr~ and Kd values for the hippocampal glutamate binding sites of near-term fetuses (GD 63) of pregnant guinea pigs that. received oral intubation of 4 g ethanol • kg-* • day-~ (n = 5 litters), isocaloric sucrose/pair-feeding (n = 5 litters), or water (n = 4 litters) from GD 2 to GD 62. The data are presented as group means + SD. There was a statistical difference (*) in the 8 ~ value for the ethanol treatment group compared with the sucrose and water treatment groups, F(2, ll) = 5.14,p < 0.05.
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group were 39°70 and 31 °70 lower, respectively, compared with the sucrose treatment group (p _< 0.05), and were 39070 and 43% lower, respectively, compared with the water treatment group (p < 0.05). There was no difference in the Kd values of the glutamate and NMDA.binding sites among the ethanol, sucrose, and water treatment groups.
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FIG. 3. Effects o f glutamate concentration on specific [3H]glutamate binding and NMDA-sensitive [3H]glutamate binding (saturation plots) in HSMP o f the near-term fetus (GD 63) o f pregnant guinea pigs that received chronic oral intubation o f 4 g ethanol • kg- i . day- i (n = 1 litter), isocaloric sucrose/pair-feeding (n -- 1 litter), or water (n = 1 litter) from GD 2 to GD 62. Curves o f best fit were obtained by computer-assisted, nonlinear regression analysis o f the data.
The overall goal of our research program is to test the hypothesis that ethanol neurobehavioural teratogenesis involving the hippocampus is due, at least in part, to a decrease in hippocampal glutamatergic function. The objective of this study was to determine the effect of chronic ethanol administration to pregnant guinea pigs on the density and affinity of glutamate and NMDA binding sites in the hippocampus of the mature, near-term fetus. The guinea pig was studied because of its extensive prenatal brain development, including the brain growth spurt. The data of the present study confirm that the selected regimen of 4 g ethanol • kg-~ • day-~ produces CNS teratogenesis without causing maternal or embryonic/ fetal lethality, as previously reported 0,7). This ethanol regimen did not change the size or gender ratio of the litter or fetal body weight. The regimen decreased brain weight com-
EFFECT OF CHRONIC M A T E R N A L E T H A N O L A D M I N I S T R A T I O N GLUTAMATE BINDING SITES 6 ¢
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FIG. 5. Bm~ and Kd values for the hippocampal NMDA binding sites of near-term fetuses (GD 63) of pregnant guinea pigs that received oral intubation of 4 g e t h a n o l • kg-m • day-m (n = 5 litters), isocaloric sucrose/pair-feeding (n = 5 litters), or water (n = 4 fitters) from GD 2 to GD 62. The data are presented as group means + SD. There was a statistical differev:ce (*) in the Bm~ value for the ethanol treatment group compared with the sucrose and water treatment groups, F(2, 11) = 6.57,p < 0.05.
pared with the isocaloric sucrose/pair-feeding and water treatment control groups, which is indicative of microencephaly. Ethanol-induced microencephaly, determined by decreased brain weight, also occurs in the neonatal rat pup following ethanol exposure during the period encompassing the peak of the brain growth spurt (postnatal day 4 to 10), with no change in body weight (6). The selective ethanol-induced microencephaly observed in the mature, near-term fetal guinea pig could be the consequence of exposure of the brain to ethanol during the brain growth spurt, which occurs prenatally in this rodent species (11). The hippocampus was selected for study because it is one of the target sites of ethanol teratogenesis. Most of the studies investigating the neurochemical, electrophysiologlcal, and morphological changes in the hippocampus, produced by prenatal ethanol exposure, have focused on the offspring in post-
381
natal life (14,19,21,24). In the rat, prenatal ethanol exposure decreases NMDA-sensitive [3H]glutamate binding in various regions of the dorsal hippocampal formation by 18% to 21%, as determined in adulthood (21). In view o f the proposed neurotrophic role of the glutamate neurotransmitter-NMDA receptor system during brain development (17), it would appear to be important to elucidate the effect o f prenatal ethanol exposure, via maternal ethanol administration, on the glutamate and NMDA binding sites in the fetal hippocampus. We studied the mature, near-term fetal guinea pig because the density of the glutamate and NMDA binding sites appears to be greater at this prenatal time of development, based on our previous ontogeny study (2). Chronic maternal ethanol administration decreased the density of the glutamate and NMDA binding sites in the fetal hippocampus compared with the control, without affecting the affinity o f these binding sites. These findings are indicative of decreased synthesis a n d / or increased catabolism of excitatory amino acid receptors, and NMDA receptors in particular, without affecting the binding characteristics of the glutamate recognition sites on these receptors. This appears to be the first reported study of the effect of prenatal ethanol exposure on fetal hippocampal glutamate and NMDA binding sites. Our results are in agreement with the reported decrease in the density of dorsal hippocampal NMDA binding sites (21) and whole-brain glutamate binding sites (16) in the adult and neonatal rat, respectively, produced by prenatal exposure to ethanol. Furthermore, in both of these studies, there was no change in the affinity of the binding sites (16,21). The decreased density of the glutamate and NMDA binding sites that we report here, together with the previously reported depressant effect of ethanol on stimulated and basal release of glutamate in the hippocampus of the mature, near-term fetal guinea pig (20), could lead to decreased function of the glutamaterglc system in this brain region, including suppression of the neurotrophic action of the glutamate neurotransmitter-NMDA receptor system. If this latter action occurs, it would be an important contributing factor in the mechanism of ethanol teratogenesis involving the hippocampus. In conclusion, chronic administration of 4 g ethanol • kgday-~ to the pregnant guinea pig throughout gestation produces microencephaly and decreased density of the hippocampal glutamate and NMDA binding sites in the mature, nearterm fetus. Further studies are required to determine whether this decreased density of fetal hippocampal glutamate and NMDA binding sites persists into postnatal fife, and to determine the relationship between the decreased density of hippocampal glutamate and NMDA binding sites and the hyperactivity and hippocampal structural injury, observed in adulthood, that result from chronic prenatal ethanol exposure. ACKNOWLEDGEMENTS
This research was supported by an operating grant from the Medical Research Council of Canada (MT-8073). S.A. was the recipient of an Eldon Boyd Fellowship (Department of Pharmacology and Toxicology) and a Queen's Graduate Award.
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