0 145-6008/8Y/1306-0776$2.00/0 ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH

Vol. 13, No. 6 November/December 1989

Cerebral Cortical Morphology and Behavior in Rats Following Acute Prenatal Ethanol Exposure

Lois A. Kotkoskie and Stata Norton

Cerebral cortical morphology and early motor development were evaluated in offspring from ethanol-exposed mothers, pairfed control mothers and ad libitum control rats. Ethanol-exposed rats received a total dose of 18 g/kg of ethanol by intubation on gestational Days 14 and 15, a critical period of development of the cerebral cortex. Pairfed control mothers received isocaloric sucrose on gestational Days 14 and 15 and were pairfed to ethanol-exposed animals from gestational Day 12 through gestational Day 20. Ethanol-exposed offspring weighed significantly less than control offspring from post- natal Day 7 through postnatal Day 28. Ethanol-exposed offspring also showed significant delays in reflex suspension (time an animal maintained its grip on a crossbar) and continuous corridor (number of turns in 5 min). The thickness of the cerebral cortex of ethanol- exposed offspring was significantly different from ad libitum and pairfed control offspring on postnatal Day 1. However, only Layer V and total cortical thickness were affected in ethanol-exposed off- spring on postnatal Day 28. The results of this study indicate that ethanol exposure during a critical period of development causes alterations in central nervous system development and develop- mental delays.

HE TERM fetal alcohol syndrome (FAS) was first T used in 1973 to describe the spectrum of effects in offspring of alcoholic mothers.' The characteristics of FAS are craniofacial dysmorphism, growth retardation, and central nervous sytem dysfunction. ' , 2 Central nervous sys- tem effects of FAS are the most severe and include: microcephaly, developmental delay, hyperactivity, and mental re ta rda t i~n .~ ,~ The intellectual deficits associated with FAS do not disappear with time and are thought to be a permanent consequence of prenatal ethanol expo- sure.'

The intellectual impairment and microcephaly of FAS in humans have been attributed to diminished brain g r o ~ t h ~ , ~ and structural brain malformation^.^^^ Animal studies of fetal alcohol effects have produced microence- phaly and reduced brain weight by administering ethanol during the early postnatal period.'.'' Additionally, chronic prenatal ethanol exposure has been reported to cause cortical thinning in ethanol-exposed offspring." Brain

From the Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas.

Received for publication November 4, 1988; revised manuscript re- ceived July 6, 1989; accepted July 6 , 1989

This research was supported in part by NS16694 and ES07079. LAK was supported by a Procter and Gamble Fellowship.

Reprint requests: Stata Norton, Ph.D. Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66103.

Copyright 0 I989 by The Research Society on Alcoholism.

malformations similar to those found in human neonates exposed to ethanol were reported in the offspring of pri- mates that were exposed to ethanol once a week during gestation. I 2 Intraperitoneal administration of ethanol on gestational Day 8 in the mouse produced gross brain malformations in the fetal mouse.I3

The results of the animal studies cited above suggest that acute ethanol exposure during critical' periods of development during gestation may alter brain develop- ment. We have previously shown that acute ethanol ex- posure during a critical period of cerebral cortex devel- opment in the rat results in brain malformations at par- turition similar to those reported in human fetuses from alcoholic mothers, namely, cerebral cortical disorganiza- tion, heterotopias, and cortical thinning. l 4

The purpose of this study was to investigate the post- natal morphological and behavioral consequences of acute ethanol exposure during gestational Days 14 and 15 in the rat. This period of gestation is at the onset of migration of neuroblasts from the subventricular zone to form the cortical plate of the cerebral cortex. The offspring of three groups, ad libitum-control, pairfed-control, and ethanol- exposed animals, were studied from birth to postnatal Day 28. Pairfed-control animals were included to control for the stress of dosing and nutritional intake of ethanol- exposed animals. Thickness of the cerebral cortex in post- natal Day 1 and postnatal Day 28 rats was used as an index of central nervous system morphology, and devel- opment of complex motor behavior was assessed using a series of developmental tests described previously. l 5

MATERIALS AND METHODS

Female, Sprague-Dawley-derived rats (CD Strain, Charles River), 225- 325 g, were housed in a temperature-controlled room with a 12-hour light/dark cycle. Females were housed with males of the same strain overnight and the morning that a vaginal smear contained sperm was counted as Day 1 of gestation. Pregnant rats were housed individually and weighed daily during gestation. On gestational Day 10, animals were randomly assigned to one of three groups: ad libitum controls ( n = 4), pairfed controls ( n = 4), or ethanol ( n = 4).

Ad libitum controls were allowed free access to Purina Laboratory Chow during the experiment and served as untreated controls. Ethanol and pairfed animals were dosed by gavage under light ether anesthesia on gestational Days 14 and 15. Ethanol animals received 4.5 g/kg ethanol (80% v/v) twice a day on gestational Days 14 and I5 for a total dose of 18 g/kg of ethanol. Pairfed rats received a sucrose solution isocaloric to ethanol twice a day on gestational Days 14 and 15. Blood ethanol levels were determined 2 hr after each dose of ethanol from 70 rl of tail blood (Sigma Kit 322-UV, Sigma Chemical Co., St. Louis, MO). Food intake

776

CEREBRAL CORTICAL MORPHOLOGY 111

of animals in the ethanol group was recorded daily from gestational Day 12 through gestational Day 20. Pairfed animals were matched in body weight to ethanol animals and were pairfed to ethanol animals from gestational Day 12 through gestational Day 20. Pairfed animals were given free access to Purina Laboratory Chow on gestational Day 21, 1 day prior to parturition. Newborn animals were weighed and sexed within 24 hr after parturition on postnatal Day 1. In order to ensure survival of ethanol-exposed offspring, only six of the largest animals (three males and three females) per litter were retained for postnatal study. Remaining offspring were used for morphometric analysis of cerebral cortex in postnatal Day 1 rats. All behavioral and morphometric analyses were conducted blind with respect to treatment group.

Postnatal Parameters

A series of developmental tests described previously15 were used to assess development of complex motor behavior of offspring in the early postnatal period.

Body Weight. Body weight was recorded on postnatal Days 7, 14, 21, and 28. All animals were identified by tattoo from subcutaneous injection of a small amount of India ink in one or more paws on postnatal Day 7.

Negative Geotaxis. Negative geotaxis was used as a test of motor development in pups daily from postnatal Day 7 through postnatal Day 14. Pups were placed on an incline of 25’ facing downward on the incline. The time required for pups to turn 180” to face upward on the incline was recorded (up to 60 sec). An average time for each pup for 8 days was computed and used for statistical analysis.

Rejlex Suspension. Reflex suspension was also measured daily in pups between postnatal Day 7 and postnatal Day 14. Animals grasped a horizontal rod suspended over a padded floor and the length of time the animal could support its own weight was measured (up to 60 sec). The longest time obtained from three consecutive trials per day was recorded. A n average time for each pup for 8 days was calculated and used for statistical analysis.

Continuous Corridor. The activity of offspring in the continuous comdor was measured on postnatal Day 28. The continuous comdor was a maze with a “Greek key” design containing 1 1 right angle turns. The number of right angle turns made by a rat in a 5-min period was recorded.

Gait. Locomotor patterns of offspring were also measured on postnatal Day 28. The hind paws of rats were dipped in ink and they were allowed to walk along a corridor 90 cm long that was covered with ink-absorbent paper. Bamers were dropped as the animals walked along the comdor to prevent backtracking. Two sets of tracks were obtained from each animal and length of at least four full strides were measured per animal. From the linear measurements of footprints, a computer program was used to calculate stride width and sine of the angle of advance for each animal.

Morphology

Morphometric analysis of cerebral cortex was performed on offspring at postnatal Day 1 and postnatal Day 28. Thickness of cortical layers were measured with a light microscope equipped with the ZIDAS image analysis system (Carl Zeiss, Inc.). With this system, microscope images were projected onto a digitizing pad. The thickness of layers was meas- ured with a cursor at the top and bottom of each cortical layer. The shortest line between the points, perpendicular to the surface of the cortex, was calculated with the ZIDAS software. Microscopic magnifi- cation was 400X for Day 1 brains and lOOX for Day 28 brains. To this was added 3x magnification from projection onto the digitizing pad. The actual distance measured with the cursor was great enough to keep measurement errors small. Pie-shaped wedges of parietal cortex in post- natal Day 1 offspring were embedded in plastic, sectioned at 2 pm and stained with toluidine blue. Cortical layers quantified in postnatal Day 1 offspring were: marginal zone, cortical plate, intermediate zone, subven- tricular, and ventricular zone, and total cortical thickness. Postnatal Day

28 animals were anesthetized with ether and perfused with Karnovsky’s fixative.16 Brains were embedded in paraffin, sectioned at 6 pm, and stained with toluidine blue. Cortical thickness was measured in frontal cortex, approximately 7 mm anterior to the external auditory meatus and 2.5 mm lateral to the midline, using stereotaxic coordinates.” Thickness of each of the six cortical layers and total cortical thickness were quantified in postnatal Day 28 offspring.

Statistics

Maternal weight gain was analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s range test. Postnatal weight gain of offspring, gait and postnatal Day 1 cortical thickness data were evaluated by two-way analysis of variance followed by Duncan’s range test. Post- natal Day 28 cortical thickness and behavioral data were not normally distributed and so were analyzed by the Mann-Whitney U Test. The alpha level for statistical significance was set at p < 0.05. Probit analysis of negative geotaxis and reflex suspension data was camed out to obtain the time at which 50% reached a preset criterion.

RESULTS

The dose of ethanol used in this study produced signs of moderate maternal ethanol intoxication without pro- ducing maternal respiratory depression. Animals were ataxic and lethargic after all doses of ethanol and were unresponsive to stimuli for 3-6 hr following the last dose of ethanol on gestational Day 15. Average maternal blood ethanol levels 2 hr after the first and second dose of ethanol on gestational Day 14 were 77 mg percent and 216 mg percent, respectively. Maternal blood ethanol levels 2 hr after each dose of ethanol on gestational Day 15 were 1 15 mg percent and 335 mg percent, respectively. All ethanol-

Gestotionol day J

Fig. 1. Maternal weight gain (mean f SE) from gestational Day 1 through gestational Day 21. N = 4 for each treatment group.

Table 1. Postoartum Parameters Treatment group

Ad libitum Pair-fed Ethanol

No. litters 4 4 4 No. live pupspitter mean (range) 16 (12-20) 14 (12-15) 16 (13-19) No. dead pups 0 1 1 Birth weight (9) mean & SE 5.7 f 0.04 6.1 t 0.08 5.5 f 0.07.

* Significantly different from pairfed controls, p < 0.05, two-way ANOVA fol- lowed by Duncan’s range test.

778 KOTKOSKIE AND NORTON

exposed animals recovered and appeared healthy after the period of ethanol intoxication.

Ad libitum control animals had a steady weight gain during gestation and gained an average of 150 g by gesta- tional Day 21, 1 day prior to parturition (Fig. 1). The weight gain of ethanol and pairfed animals was altered during the second half of gestation. Ethanol-exposed ani- mals had a significantly reduced weight gain compared with ad libitum control animals from gestational Days 16 through 19. Pairfed control animals had a significantly decreased weight gain from gestational Days 15 through 21. In summary, pairfed control animals and ethanol- exposed animals had a similar pattern of weight gain during gestation, but the depression of maternal weight gain was more prolonged in pairfed control rats than ethanol-exposed rats.

Although maternal weight gain was depressed in pairfed and ethanol-exposed animals, birth weight of ethanol- exposed offspring was significantly lower than pairfed offspring (Table 1). The birth weights of ad libitum and pairfed were not significantly different. Acute prenatal ethanol exposure did not alter litter size or neonatal mor- tality. However, the growth rate of ethanol-exposed off- spring was significantly different from both ad libitum and pairfed controls through postnatal Day 28 (Fig. 2).

Behavior Offspring in the ethanol group showed delays in motor

development as measured by decreased reflex suspension time and decreased continuous corridor activity (Table 2). Performance in the negative geotaxis test was unaffected by acute prenatal ethanol exposure. Pairfed control ani- mals showed a significant increase in reflex suspension time compared with ad libitum control and ethanol off- spring. The diminished reflex suspension time in ethanol- treated animals was due to a developmental delay. When time to criterion was used as a measure of performance, the time for 50% to sustain their weight for 12 sec was

t

l o t

0-0 A-A 0--0

0

0

Ad Lib Pairfed Ethanol

B 04 i 0 7 14 21 28

Postnatal Day

Fig. 2. Offspring growth rate from postnatal Day 7 to 28. Values shown are mean of 24 animals from four litters per treatment group. Ethanol-exposed animals significantly different from pairfed and ad libitum controls by two-way ANOVA, f(287.2)=96,0, p < 0.0001, Maximum standard error for points on the graph was *2 g.

11.2 days, compared with 10.2 days for ad libitum and 8.4 days for pairfed animals (Fig. 3). Gait showed no significant differences among any of the treatment groups (Table 3).

Morphology Ethanol-exposed offspring had significantly thinner cor-

tices than ad libitum and pairfed control offspring on postnatal Day 1 (Table 4). Of the four cortical layers, the intermediate zone, subventricular and ventricular zones were most affected by prenatal ethanol exposure. Cortical plate thickness of ethanol-exposed offspring on postnatal Day 1 was significantly different only from pairfed control offspring. The marginal zone of postnatal Day 1 cerebral cortex was not significantly affected by acute prenatal ethanol exposure. There were no significant differences in the thickness of any cortical layer between ad libitum and pairfed offspring on postnatal Day 1.

The architecture of the cerebral cortex of ethanol-ex- posed offspring on postnatal Day 28 did not show major alterations from acute gestational exposure to ethanol. However, the average thickness of frontal cortex in post- natal Day 28 animals was reduced. In addition, ethanol- exposed offspring had some disorganization of cortical layering, particularly noticeable as patches where different layers were irregular in thickness (Fig. 4). Lamina V and total cortical thickness were thinned following acute pre- natal ethanol exposure. Lamina V was significantly differ- ent in ethanol-exposed offspring compared with ad libitum control offspring. Ethanol-exposed offspring also had sig- nificantly thinner cortices than pairfed control offspring on postnatal Day 28. There were no significant differences in the thickness of any cortical layer between ad libitum and pairfed control offspring on postnatal Day 28 (Table 5).

DISCUSSION

Acute ethanol exposure on gestational Days 14 and 15 produced significant maternal intoxication. The maternal blood ethanol levels measured in this study were not associated with maternal respiratory depression, so the effects of ethanol on the fetus were not confounded by maternal hypoxia. Ethanol-exposed animals decreased their food intake and lost weight on gestational Days 15 and 16. Food intake may have been decreased because of gastritis and appetite depression associated with ethanol consumption.

The reduced birth weight of ethanol-exposed offspring was not the result of maternal undernutrition and de- pressed weight gain during gestation. Pairfed animals had a similar food intake and weight gain during gestation and yet their pups were heavier at birth than ethanol-exposed offspring. However, pairfeeding does not control for the altered absorption, metabolism or excretion of nutrients that may occur following ethanol exposure. For example,

CEREBRAL CORTICAL MORPHOLOGY 779

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Table 2. Behavioral Data

Treatment group'

Ad libitum Pairfed Ethanol

Average Days to Average Days to Average Days to performance7 criterion* performance criterion performance criterion

Negative geotaxis (sec to 21.9 f 1.4 10.1 f 0.5 22.3 f 1.4 10.0 f 0.4 21.1 f 1.3 9.8 f 0.4

Reflex suspension (sec) 11.9 -C 0.7 10.2 f 0.4 17.7 f 1.45 8.4 ir 0.4s 8.4 f 0.8&1 11.2 f 0.45.1 Continuous corridor (no. 40.8 f 1.8 39.1 f 1.7 30.1 f 1.45,ll

turn)

turns)

* n = 24 (4) for each treatment group. t Average for all tests. $ Time at which 50% reached criterion (negative geotaxis = turning in 10 sec; reflex suspension = suspension for 12 or more sec) with 95% limits, probit analysis. 5 Significantly different from ad libitum controls, p -= 0.002, Mann-Whitney U Test. T Significantly different from pair-fed controls, p -= 0.0001, Mann-Whitney U Test.

/ I R-0 7 0 9 10 11 12 13 14

DAY

Fig. 3. Change in reflex suspension behavior from postnatal Day 7 to 14 based on the percentage of animals achieving the criterion of sustaining their weight for 12 sec or more. Symbols: X = pairfed, 0 = ad libitum, 0 = ethanol.

Table 3. Gait Analysis

Treatment group' _____

Ad libitum Pairfed Ethanol

Gait length (mm) 101.4 f 1.8 99.3 f 1.7 99.1 k 2.2 Gait width (mm) 29.6 -C 0.6 31.5 f 0.7 28.3 f 0.6 Sine of alternation 0.51 2 0.01 0.52 f 0.01 0.51 C 0.01

+ All values expressed as mean C SE. N = 24 (4) for each treatment group.

Table 4. Postnatal Day 1 Cortical Thickness (pm)

Treatment group'

Cortical layer Ad libitum Pairfed Ethanol

Marginal zone 60 f 3 59 f 3 53 f 5

Intermediate zone 347 k 16 334 f 8 287 f 9t,* Cortical plate 6 9 8 f 1 6 7 3 6 f 1 5 6 8 0 f 1 8 t

Subventricular and ven- 147 f 7 138 f 4 117 f 8t,*

Total thickness 1259 f 23 1272 f 22 1139 f 26t.S tricular zone

* All values expressed as mean 2 SE. N = 16 (4) for each treatment group. t Significantly different from pairfed controls. p -= 0.05, two-way ANOVA fol-

* Significantly different from ad libitum controls, p < 0.05, two-way ANOVA lowed by Duncan's range test.

followed by Duncan's range test.

ethanol exposure in the rat reduces intestinal'* and placental" transport of zinc, an essential nutrient for fetal growth. Reduced nutrient availability to the fetus during the period of ethanol intoxication might have contributed

to the light birth weight observed in ethanol-exposed off- spring.

The growth rate of offspring exposed prenatally to ethanol was reduced through postnatal Day 28 and no catch-up growth occurred. Growth retardation of ethanol- exposed offspring might have resulted from poor lacta- tional performance, because ethanol inhibits the milk ejection reflex.20-21 However, it is improbable that acute ethanol exposure on gestational Days 14 and 15 would affect lactation throughout the entire postnatal period. Alternatively, altered sucking behavior of ethanol-exposed offspring might contribute to the growth retardation ob- served in this study. Delayed development of sucking behavior has been reported in offspring of rats following ethanol exposure on gestational Day 8.22 Sucking pressure, a measurement of milk intake, was significantly decreased in offspring of women who consumed two or more drinks per day during pregnancy.23 Therefore, even though lac- tational performance was probably satisfactory, ethanol exposed offspring may have been unable to nurse properly and so had a reduced growth rate compared with ad libitum and pairfed control offspring.

A series of behavioral tests was used in this study to assess development of complex motor behaviors of off- spring during the early postnatal period. Delayed appear- ance of a behavior often reflects uncompensated damage to the developing nervous system. In the present study, delayed development of complex motor behavior was noted in ethanol-exposed offspring. Reflex suspension per- formance was delayed. However, the other preweaning test of motor development, negative geotaxis, was not altered by exposure; on gestational Days 14 and 15. In a previous study, a delay in both negative geotaxis and reflex suspension time was reported following chronic prenatal ethanol exposure from gestational Day 5 to postnatal Day 10." The difference in results with the negative geotaxis test in the two studies may reflect a lack of sensitivity of the critical components to ethanol on gestational Days 14 and 15. Continuous corridor activity on postnatal Day 28 was also decreased in ethanol-exposed offspring. Contin- uous corridor is a test that involves at least two compo- nents: (1) locomotion, and (2) the animals's response to a

780 KOTKOSKIE AND NORTON

Table 5. Postnatal Day 28 Cortical Thickness (pm)

Treatment group’

Cortical layer Ad libitum Pairfed Ethanol

I 134 (81-214) 152 (106-190) 132 (92-202) I 1 291 (207-357) 302 (165-441) 279 (173-452) 111 31 2 (1 84-472) 334 (21 4-482) 335 (1 79-460) IV 91 (36-173) 78 (48-165) 84 (48-167) V 442 (288-575) 416 (298-631) 380t (253-571) VI 500 (345-771) 551 (405-717) 532 (369-817) Total 1762 (1380-2070) 1833 (1618-2023) 1741$ (1415-1999)

All values expressed as mean (range). N = 24 (4) for each treatment group. t Significantly different from ad libitum controls, p < 0.02, Mann-Whitney U

* Significantly different from pair-fed controls, p c 0.02, Mann-Whitney U Test. Test.

novel environment. One possibility was that acute prenatal ethanol exposure decreased continuous corridor activity because ethanol-exposed offspring did not have normal motor function and hence did not explore the maze. However, gait data, also measured on postnatal Day 28, was not different from controls. Because ethanol-exposed offspring had normal gait, altered motor function did not account for the results obtained in this study. The second possibility is related to the characteristics of exploratory activity in young rats. It has previously been shown that the amount of continuous corridor activity increases after weaning. Postnatal Day 21 control animals have a low continuous comdor activity compared with postnatal Day 28 and adult control animals.” It is possible that the expected increase in continuous corridor activity between postnatal Day 21 and 28 was delayed in ethanol-exposed offspring.

Although the largest and presumably most normal off- spring from ethanol-exposed mothers were studied post- natally, the animals differed from controls in growth, behavior and cortical morphology.

Fig. 4. Comparison of cortex of pairfed (A) and ethanol-treated (8) rats on postnatal Day 28. Ethanol-treated cortex is thinner and cells of cortical columns are not in organized vertical rows. Marginal zone shows wavy orientation. Toluidine blue stain. Bar = 200 pm.

The major effect on the frontal area of the cerebral cortex was that the cortex was thinner in ethanol-exposed rats. On postnatal Day 28, the most notable thinning was in Layer V. In a previous report, using similar measure- ments but with dietary exposure to ethanol, thinning of the cortex was also reported.” The most marked effect on the cortex in that study was in Layer VI, possibly reflecting the prolonged and late exposure to ethanol. It has been reported that ethanol reduces the number of neurons generated in fetuses exposed on gestational Days 6 to 2 1 .24

Although it is possible that differences in behavioral out- come are related to differences in effect on the cerebral cortex during the period of formation, the causal relation- ship is uncertain.

It has been previously reported that severe brain mal- formations are present in some fetuses on gestational Day 2 1 following ethanol exposure on gestational Days 14 and 15.14 Some disorganization of cortical layering was found in ethanol-exposed offspring on postnatal Day 28 but severe malformations were not evident. There are two reasons for the lack of severe cortical malformations in these ethanol-exposed offspring on postnatal Day 28. Only the six largest animals at birth were retained for postnatal study. It is possible that offspring with low birth weight had the most severe brain malformations and so were not retained for further study. Secondly, it has been shown by several investigators that cortical damage caused by pre- natal exposure to toxic agents becomes less severe with mat~ration.~’-~’ Abnormalities of cytoarchitecture in the cerebral cortex of mice exposed to cytosine arabinoside on gestational Days 14 and 15 were very severe at birth, but became less distinct by postnatal Day 30 and were still accompanied by cortical thinning.25 It was hypothesized that the great increase in cortical surface area and dendritic

CEREBRAL CORTICAL MORPHOLOGY 78 1

branching of cortical neurons after parturition modified the cortical abn~rmalit ies.~~ Repair of brain malforma- tions in rats following in utero exposure to x-irradiation has been documented in several s t u d i e ~ . ~ ~ , ~ ’ Exposure to 200 r on gestational Day 13 in the rat immediately pro- duces massive cell death within the forebrain. During repair, rosettes of ventricular zone cells form within the developing cerebral cortex and are prominent at birth. With maturation, the rosettes gradually resolve, leaving behind a thinned cerebral cortex with disorganized layers in the adult A similar result has been reported with a lower dose of radiati~n.~’ The absence of cortical malformations in ethanol-exposed offspring on postnatal Day 28 supports other findings in this area of research.

The question remains as to whether or not cortical thinning on postnatal Day 28 is a permanent morphologic effect of acute prenatal ethanol exposure. Neuronal prolif- eration and migration within the cerebral cortex of the rat are complete by postnatal Day 628 and neuropil develop- ment, measured by dendritic branching and axonal den- sity, is complete by postnatal Day 30 in the rat.29 There- fore, the cerebral cortex of the rat on postnatal Day 28 is well developed and further recovery is not expected.

An epidemiology study has reported that pregnant al- coholic women increase the number of binge drinking episodes during pregnan~y.~’ Additionally, binge drinking during gestation has been associated with brain malfor- mations in human neonates8 This experiment adds to the evidence that acute ethanol exposure during a critical period of development may cause some of the manifesta- tions of FAS, such as growth retardation and delayed motor development. The importance of binge drinking in the pathogenesis of FAS needs further confirmation in human and animal studies.

ACKNOWLEDGMENTS

The expert technical assistance provided by Annette Chrzanowski and Rebecca Atherton is gratefully acknowledged.

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