Prenatal ethanol and stress in mice: 2. Development and behavior of fostered offspring

Physiology & Behavior,Vol. 45, pp. 541-549. © Pergamon Press plc, 1989. Printed in the U.S.A.

0031-9384/89 $3.00 + .00

Prenatal Ethanol and Stress in Mice: 2. Development and Behavior of Fostered Offspring G . R. W A R D * A N D P . E . W A I N W R I G H T t

1

*Department o f Psychology, tDepartment o f Health Studies, University o f Waterloo Waterloo, Ontario, N2L 3G1, Canada R e c e i v e d 19 M a y 1988 WARD, G. R. AND P. E. WAINWRIGHT. Prenatal ethanol and stress in mice: 2. Development and behavior of fostered offspring. PHYSIOL BEHAV 45(3) 541-549, 1989. - O n days 12 to 17 of gestation, B6D2F2 mice were pair-fed liquid diets containing either 25% ethanol-derived calories or an isocaloric amount of maltose-dextrin. During this time, half the mice in each dietary condition also underwent two daily one-hour periods of restraint stress. All pups were fostered at birth to untreated mothers whose pups, in turn, were fostered to the treated mothers. Two additional groups of untreated dams were included, the pups in one group being fostered to other untreated dams while the rest remained with their biological mothers. Prenatal ethanol retarded neurobehavioral development on day 32 postconception and also decreased pup body weight at birth and at weaning, and brain weight on day 32 and at weaning. Prenatal stress decreased body weight at birth in the pair-fed controls only, increased open field activity at weaning and affected retention of a learned passive avoidance task. Undernutrition due to the pair feeding procedure decreased pup birth weight. There were a few effects on untreated pups fostered to treated dams, but these were marginal. These results do not support a role of stress-induced physiological changes in ethanol teratogenicity. Prenatal ethanol Prenatal stress Neurobehavioral development Activity Fostering Mice Maternal effects

E T H A N O L consumption has been reported to increase plasma corticosterone concentrations (PCC) in pregnant rats (21), and high P C C during pregnancy have been implicated in abnormal morphological and behavioral development in offspring (2,3). These findings have important implications for research into the effects of prenatal ethanol exposure. We have found that restraint stress in pregnant mice, which is capable of elevating P C C to a much greater extent than is ethanol consumption alone, led to an amelioration of the prenatal ethanol-induced delay in neurobehavioral development in preweanling offspring (19). Moreover, we found that stress significantly reduced the blood alcohol concentrations (BAC) in the pregnant dams, suggesting that, under some circumstances, the physiological changes triggered by stress may serve a protective function. In this experiment, our purpose was to replicate the effect on behavioral development, as well as to investigate the combined effects o f ethanol and stress during pregnancy on brain development and on behavior in the weaning-aged offspring. In order to separate the prenatal effects of our treatments from postnatal effects due to alterations in maternal behavior and lactational performance, we employed a surrogate fostering design in which all pups of treated dams were fostered after birth to untreated control dams, while the pups of these untreated dams were themselves reared by the treated dams.

Shock avoidance

Brain development

We have found that mice show reductions in brain weight following maternal exposure to a diet containing 25% ethanolderived calories (18). Because there is evidence that brain growth is less sensitive to lower doses of ethanol (20), we would predict that, by lowering BAC, restraint stress might alleviate ethanol-induced effects on brain weight. Furthermore, several authors have reported that hyperactivity (5,23) and, more specifically, deficits in response inhibition (4, 7, 11) can result from prenatal ethanol exposure. Interestingly, prenatal stress has also been reported to cause increased l o c o m o t o r activity (10,14), although most studies report decreased activity in offspring (6, 8, 12), or no change in activity levels (9,13). Therefore, we examined the effects of these prenatal treatments on locomotor behavior and on passive shock avoidance learning at weaning. METHOD

Subjects The parental mice were male and primiparous female B6D2F~ mice purchased from Charles River Breeding Laboratories, St. Constant, Quebec when they were four weeks of age. They were housed in groups of three to five animals o f the same sex in standard opaque plastic mouse cages (29x 18x 13

I Requests for reprints should be addressed to P. E. Wainwright.

541

542 cm) with Beta-Chip hardwood bedding and several sheets of toilet tissue for nesting material, and were maintained under a reversed 12-hour dark:light cycle (dark 0800-2000 hr). All mice were provided with free access to laboratory chow (Master MLM rodent food, Maple Leaf Mills, Toronto) and tap water until the time of mating at between 19 and 29 weeks of age.

Experimental Design The experimental design was a 2x2 factorial design, with two levels of stress (restraint or no restraint) and two levels of ethanol-derived calories (EDC) (25070 EDC or 0070 EDC) in a Sustacal chocolate-flavored diet, with the 007o diet containing maltose-dextrin substituted isocalorically for ethanol. The formulation of the liquid diets has been described previously (19). All pregnant females were matched on the basis of body weight on the day of conception (day 0) as well as on day 12 of gestation, and pair-fed during treatment to their weight-matched partner in the group receiving both the ethanol and stress treatments. On day 20 postconception, which is approximately 24 hours after birth, all offspring of treated dams (i.e., the treated pups) were fostered to dams which had been allowed free access to lab chow and water throughout pregnancy and left undisturbed except for routine cage changing. Conversely, the offspring of these untreated dams (i.e., the untreated pups) were fostered to the treated dams. In all cases, fostering took place between dams which had been mated on the same day. In addition to the four groups of treated pups and the four groups of untreated pups, two additional untreated groups were included. The pups in one of these groups were fostered on day 20 to other untreated dams while the pups in the other group were reared by their biological mothers. By providing a comparison with the pups in the unstressed maltose-dextrin group, the pups of the fostered lab chow control group allowed for the assessment of inadequate nutrition in the groups receiving the liquid diets. Comparing the fostered pups of the control dams with those of the nonfostered controls allowed the effects of the fostering procedure to be assessed, and the nonfostered control pups provided a control for testing the reliability of the behavioral scale. Within each of the two fostering conditions, the four experimental groups were designated as follows (n=No. of litters of treated pups at birth: slight discrepancies between these figures and those reported in the tables reflect missing data due to random attrition): Ethanol/Stress (E/S) (n= 15) Ethanol/No Stress (E/NS) (n=16) Maltose-Dextrin/Stress (MD/S) (n = 15) Maltose-Dextrin/No Stress (MD/NS) (n= 14) The two untreated control groups were designated as follows: Lab Chow/Fostered (LC/F) (n= 10) Lab Chow/Not Fostered (LC/NF) (n=9)

Procedure The general procedures pertaining to the treatment of the pregnant dams were described in detail previously (19). Briefly, pregnant females were housed individually on the day of mating (day 0) and were left undisturbed until day 10 when their lab chow and water were replaced with the 0o7o EDC liquid diet. They were fed 0.625 kcal/g body weight of this diet on days 10 and 11 and, on day 12, each female was assigned to a treatment group on the basis of body weight and fed the appropriate diet. The liquid diets were replenished each morning at the beginning of the dark cycle, and the E/S group was allowed

WARD AND WAINWRIGHT to feed ad lib while all other groups were pair-fed on an individual basis to the animals in this group. On day 17, the ethanol content of the diet was reduced to 10°70 EDC to alleviate possible withdrawal effects and, on day 18, all animals were returned to lab chow and water ad lib. The stressing procedure consisted of restraining the mouse by stapling a piece of wire mesh screening around it tightly enough to restrict movement. All stressed mice were restrained for one hour twice daily beginning at 1030 hr and again at 1730 hr from days 12 to 17, while all unstressed mice receiving liquid diets had their diets removed during each of these one-hour periods. All treated dams were weighed on day 0 and then daily from days 12 to 17 while all untreated dams (i.e., foster mothers as well as those in the lab chow control groups) were weighed on days 0, 12 and 17 and otherwise left undisturbed. At noon on day 20, all pups were weighed and each litter was randomly culled to eight and assigned to its appropriate foster mother, except for the LC/NF litters which remained with their biological mothers. On day 32, all pups were weighed and two pups of each sex were randomly selected to undergo a battery of sensorimotor tests designed to assess behavioral development. This battery was developed by Wahlsten (15,16) and has been described in detail previously (17). Briefly, each pup was scored on eleven tasks involving righting, grasping, climbing and cliff avoidance, as well as visual and auditory awareness. The mean score of all tasks was averaged over the litter and converted into the mean behavioral age, in days, of that litter by using the quadratic equation y=24.40 + 11.14x - 1.09x 2. This equation was derived from normal B6D2F2 litters tested on each of days 2736. After the behavioral testing, one pup of each sex was given an overdose of sodium pentobarbital and perfused intracardially with 10% buffered formalin. The brain was then removed, stored in 1007o buffered formalin for one week, the brain stem and olfactory bulbs were removed, and the brain was weighed. On day 50, all pups again were weighed and one pup of each sex, which did not undergo testing on day 32, was randomly selected to undergo a measure of activity combined with a passive shock avoidance test. Testing was done in a twochambered Plexiglas box (33 x 16.5 x 10 cm) divided in the middle by a partition. The floor of one chamber consisted of solid Plexiglas (the no-shock compartment) while the floor of the shock compartment was a grid consisting of stainless steel bars, 1.5 mm in diameter, 1 cm apart. Connecting the chambers was a 5.0x5.0 cm opening which could be closed by means of a sliding door. The avoidance task, which was conducted under red light, consisted of one session on each of two consecutive days. In the first session, a shock generator delivered 0.1 mA of continuous shock to the grid floor of the shock compartment. The mouse was placed into the no-shock compartment with the sliding door closed. After 120 seconds, the door was opened and the session continued for another 300 seconds or until the mouse stayed out of the shock compartment for 120 seconds following its most recent entry. The behaviors recorded were the latency to first shock and the total number of shocks received. In the second session, 24 hours later, the mouse again was placed into the no-shock compartment for 120 seconds with the door closed. This time, however, when the door was opened, no shock was delivered to the grid, and the second phase lasted until the mouse entered the second compartment, or until 300 seconds had elapsed. The behaviors recorded in the second session were the latency to the first entry in which two paws were placed on two separate bars (which would have produced a shock had the shock generator been on). Activity was measured in each session during the time in which the mouse

ETHANOL, STRESS AND FOSTERING

543 TABLE 1

EFFECTS ON MATERNAL AND PUP WEIGHT OF ETHANOL EXPOSURE COMBINED WITH RESTRAINT STRESS ON DAYS 12-17 POSTCONCEPTION* (ALL TREATED PUPS FOSTERED TO UNTREATED DAMS)

Ethanol

Maternal Weight (g) Day 0

Day 12

Day 17

Litter Size

Pup Weight (g) Day 20 (Birth)

Day 32

Day 50 (Weaning)

Malt-Dex

Lab Chow

Stress

No Stress

Stress

No Stress

Control

25.5 I0.5] (15) 30.9 [0.6]

25.7 [0.3] (16) 31.3 [0.4]

26.5 [0.5] (15) 31.6 [0.5]

25.9 [0.31 (14) 31.4 [0.4]

25.6 [0.3] (19) 30.9 [0.5]

(15)

(16)

(15)

(14)

(19)

37.2 [0.8]

36.3 [0.5]

36.9 [0.7]

36.2 [0.8]

42.5 [0.81

(15)

(16)

(15)

(14)

(19)

8.9 [0.6] (15)

10.0 [0.4] (16)

9.9 [0.5] (15)

9.7 [0.6] (14)

9.2 [0.5] (19)

1.20 [0.01] (12)

1.18 [0.03l (13)

1.23 [0.031 (13)

1.38 [0.02] (10)

1.49 [0.021 (9)

6.43 [0.19] (13) 16.89 [0.431 (13)

6.59 [0.18] (15) 17.02 [0.331 (14)

6.55 [0.18] (14) 17.45 [0.521 (14)

6.74 [0.171 (13) 18.49 [0.43] (10)

7.12 [0.19] (8) 18.52 [0.57] (8)

Effectst

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A§

*Data are presented as means with [SEM], and (n)=number of litters. IA Main effect of Ethanol; B Main effect of Stress; AxB Interaction; C Nutritional effect. :~p
was in the no-shock compartment with the door closed. The floor of the compartment was divided into four squares of equal size by means of two bisecting lines, and the number of times all four paws of the mouse crossed from one square into another during the 120 second trial determined the locomotion score. As well, the number of times that the mouse reared up on its hind legs was recorded. All testing throughout the study was conducted with the experimenter unaware of the treatment condition of the mouse. The two animals were then killed and their brains removed and weighed as described above.

Nutritional effect: L C / F vs. MD/NS. Fostering effect: LC/F vs. LC/NF. Preliminary analysis of the data included sex as a variable and, in the absence of a significant sex by treatment interaction, further analyses were conducted on the data collapsed across sex using the litter mean score as the unit of analysis. A repeated measures analysis was done on the individual day 50 behavioral scores with separate analyses for pup and maternal effects. Alpha was set at p=0.05. RESULTS

Data Analysis The data were analysed using the general linear model (GLM) provided by the Statistical Analysis System (SAS) to do analysis of variance (ANOVA) and covariance (ANCOVA). The experimental hypotheses, which are listed below, were addressed by preplanned contrasts, as described fully in our previous study (19). The contrasts were carried out within each of the subsets of the data, i.e., treated pups reared by untreated dams and untreated pups reared by treated dams. Main effect of ethanol: E/S + E/NS vs. MD/S + MD/NS. Main effect of stress: E/S + MD/S vs. E/NS + MD/NS. Ethanol by Stress interaction: E/S + MD/NS vs. E/NS + MD/S.

Maternal Variables The data on the treated mothers are shown in Table 1. On day 17 of gestation there was a significant nutritional effect with the MD/NS dams being lighter than the LC mothers, F(1,74)=37.70, p<0.0001. There was no significant effect on litter size, F(4,74)=0.80, p>0.05. The percentage of each day's food allotment which was still uneaten was recorded each day after 8 hours of feeding and the group means are shown in Fig. 1. Repeated measures analysis showed significant group differences in the amount remaining after 8 hours, F(3,56)= 47.79, p<0.0001, as well as a significant increase in this amount over the 6 days of the treatment, F(5,261)=12.69, p<0.0001.

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FIG. 1. Effect of ethanol and restraint stress on the percentage of the total daily food remaining after 8 hours of feeding in pregnant dams. Ethanol p<0.05; Stress .o<0.05.

Two-way analysis of variance on each day of treatment showed significant main effects of both ethanol and stress leading to reductions in the amount of food consumed in the first 8 hours, and these effects were significant on every day of treatment.

Pup Effects." Treated Pups Fostered to Untreated Dams Body weight. The data on pup body weight presented in Table 1 show a significant main effect of ethanol in reducing pup weight on day 20, F(1,108)=14.91, p<0.001, as well as an interaction of ethanol and stress, with stress causing a reduction in weight only in the MD condition, F(1,108)=8.47, p<0.01. There was also a nutritional effect due to pair-feeding leading to reduced day 20 weights in the MD/NS group compared to the LC/F group, F(1,108)=6.34, p<0.02. There was no significant effect on weight on day 32 but, on day 50, ethanol-treated animals were significantly lighter in weight, F(1,103)=6.01, p<0.02. Behavioral development. The data on behavioral development on day 32 are shown in Fig. 2. They show a main effect of ethanol in causing a retardation of 0.5 days, F(1,111)= 15.30, p<0.001, but none of the other effects was significant. Brain development. Brain weights on both day 32 and day 50, as shown in Figs. 3 and 4, were significantly reduced by ethanol [day 32, F(I,111)=7.66, p<0.01, day 50, F(1,102)=8.17, p<0.01]. In addition, on day 32, there was a nutritional effect with the MD/NS group having lighter brains than the LC group, F(I,I 11)=3.59, p<0.05 (one-tailed). Analysis of covariance showed that the ethanol effect was independent of the effect on body weight on both day 32, F(1,110)=5.75, p<0.02, and day 50, F(1,101)=4.21, p<0.05, but there was no longer a significant nutritional effect on brain weight on day 32 when adjusted for differences in body weight. Because of a significant group by sex interaction for day 50 brain weight, F(9,194)=2.04,

p<0.05, separate analyses were conducted for each sex. For females, there were still significant main effects for ethanol, F(1 36)=3.50, p<0.05 (one-tailed), and for nutrition F(1,96)= 8.70, p<0.005. For males, there was no nutritional effect but there was still a main effect for ethanol, F(1,98)=3.40, p<0.05 (one-tailed). Activity and passive avoidance learning. Locomotion and rearing scores were summed to obtain overall general activity scores for each pup. Since analyses of each of these separate categories yielded identical results, only the activity scores, as shown in Fig. 5, are presented here. Repeated measures analysis showed a decrease in activity over days, F(1,122)= 17.32, p<0.0001, as well as the stressed animals being more active on each of the days, F(5,129)=4.35, p<0.001. All groups learned to inhibit entry into the shock compartment from day 1 to day 2, F(1,114)=139.82, p<0.0001 (Fig. 6). On the first day, the latency did not differ among groups but, on the second day, the stressed animals entered sooner, F(5,121)=2.45, p<0.05. There was no ethanol effect on either activity or latency.

Maternal Effects." Untreated Pups Fostered to Treated Dams These data are shown in Tables 2 and 3. The only significant effects were that the pups reared by ethanol-treated dams weighed less on day 32, F(1,111)=3.44, p<0.03 (one-tailed), and had lighter brains on day 50, F(1,102)=3.46, p<0.03 (onetailed), and when brain weight analyses were conducted on each sex individually, the ethanol effect was present only in the males. As well, pups reared by stressed dams weighed less on day 50, F(1,103)=5.48, p<0.02. The fostering procedure had a detrimental effect on day 32 body weight, F(1,111)=3.39, p<0.05 (one-tailed) and behavioral development, F(I, 111)=3.52, p<0.05 (one-tailed), and on day 50 brain weight in the female offspring only, F(1,96)=5.99, p<0.02.

E T H A N O L , STRESS A N D F O S T E R I N G

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FIG. 2. Effect of prenatal ethanol combined with restraint stress on the behavioral development of B6D2F2 mice on day 32 postconception. All pups were reared by untreated dams. Data are collapsed across sex and values are group means _+ SEM, based on litter mean scores. Ethanol p<0.001.

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ETHANOL MALT--DEX LAB CHOW PRENATAL TREATMENT

FIG. 3. Effect of prenatal ethanol combined with restraint stress on the brain weight of B6D2F2 mice on day 32 postconception. All pups were reared by untreated dams. Data are collapsed across sex and values are group means + SEM, based on litter mean scores. Ethanol p<0.05; Nutrition p<0.05 (one-tailed).

DISCUSSION

In this study, prenatal ethanol exposure led to reduced body weights in the offspring at birth as well as at weaning, and reduced brain weights on day 32 p o s t c o n c e p t i o n a n d at wean-

ing. F u r t h e r m o r e , behavioral d e v e l o p m e n t at day 32 was significantly retarded due to the ethanol treatment, but there were no main effects o f ethanol on either locomotor activity or passive avoidance learning at weaning. On the other hand, prenatal stress had no effect on any o f the morphological measures, or

546

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FIG. 4. Effect of prenatal ethanol combined with restraint stress on the brain weight of B6D2F2 mice on day 50 postconception. All pups were reared by untreated dams. Data are collapsed across sex and values are group means _+ SEM, based on litter mean scores. Ethanol p<0.01.

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FIG. 5. Effect of prenatal ethanol combined with restraint stress on activity of B6D2F2 mice at weaning. All pups were reared by untreated dams. Data are collapsed across sex and values are group means + SEM, based on one animal of each sex in each litter. Time p<0.0001; Stress

p<0.001.

on day 32 behavioral development, but did increase locomotor activity, as well as affect the retention o f a passive avoidance response at weaning. The fact that these effects were present in offspring that were reared by untreated foster mothers indicates

that the effects were due to the prenatal treatments rather than to any postnatal maternal differences caused by the treatments. Effects o f ethanol consumption by pregnant dams m a y have continued postnatally since prenatally-untreated pups reared

ETHANOL,

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FIG. 6. Effect of prenatal ethanol combined with restraint stress on passive avoidance learning of B6D2F 2 mice. All pups were reared by untreated dams. Data are collapsed across sex and values are group means _+ SEM, based on one animal of each sex in each litter. Time p<0.0001; Stress (Day 2) p<0.05.

TABLE 2 MATERNAL EFFECTS ON MOUSE PUP DEVELOPMENT OF ETHANOL EXPOSURE COMBINED WITH RESTRAINT STRESS ON DAYS 12-17 POSTCONCEPTION a (ALL UNTREATED PUPS FOSTERED TO TREATED DAMS) Ethanol

Body Weight (g) Day 20

Day 32

Day 50

Brain Weight (g) Day 32

Day 50

Developmental Age (Days) Day 32

Malt-Dex

Lab Chow

Stress

No Stress

Stress

No Stress

Fostered

Nonfostered

1.50 [0.02] (12) 6.67 [0.19] (11) 17.78 [0.43] (14)

1.47 [0.02] (13) 6.97 [0.11] (13) 18.38 [0.38] (12)

1.53 [0.04] (14) 7.06 [0.21] (10) 17.67 [0.43] (7)

1.49 [0.04] (10) 7.29 [0.12] (12) 19.26 [0.39] (9)

1.49 [0.02] (9) 7.12 [0.19] (8) 18.51 [0.57] (8)

1.45 [0.03] (12) 6.58 [0.25] (12) 17.93 [0.361 (12)

0.4045 [0.0050] (11) 0.4413 [0.0032] (14)

0.4042 [0.0035] (13) 0.4327 [0.0049] (11)

0.4048 [0.0050] (10) 0.4484 [0.0053] (7)

0.4103 [0.0037] (12) 0.4434 [0.0043] (9)

0.4096 [0.0050] (8) 0.4449 [0.0034] (8)

0.3957 [0.0048] (12) 0.4375 [0.0060] (12)

31.97 [0.O7] (11)

31.80 [0.12] (13)

31.86 [0.13] (10)

31.87 [0.11] (12)

31.93 [0.081 (8)

31.58 [0.17] (12)

aData are presented as means with [SEM] and (n)=number of litters. hA: Main effect of Ethanol; B: Main effect of Stress; A x B : Interaction; C: Nutritional effect; *p<0.05. cOne-tailed test.

EFF b

A *c

B*

A *c

548

WARD AND WAINWRIGHT

TABLE 3 MATERNAL EFFECTS ON BEHAVIOROF WEANLING MICE OF ETHANOL EXPOSURE COMBINED WITH RESTRAINT STRESS ON DAYS 12-17 POSTCONCEPTIONa (ALL UNTREATED PUPS FOSTERED TO TREATED DAMS) Ethanol

Activity Day 1

Day 2

Entry Latency (sec) Day 1

Day 2

Malt-Dex

Lab Chow

Stress

No Stress

Stress

No Stress

Fostered

Nonfostered

EFF b

42.1 [3.1] (28) 29.5 I2.4] (28)

35.0 [3.51 (21) 32.8 [2.2] (20)

33.4 [2.5] (14) 29.1 13.0] (14)

39.9 [3.21 (16) 28.8 [3.0] (16)

34.6 [4.2] (14) 29.1 [3.2] (12)

39.3 [3.31 (24) 30.5 I2.91 (22)

AxB*

23.5 [4.1] (27) 130.6 [22.1] (27)

27.9 [5.01 (19) 153.4 I26.31 (18)

30.8 [10.3] (13) 148.5 [35.9] (11)

29.8 [4.21 (15) 153.9 [26.9] (15)

38.3 [10.71 (14) 165.4 [33.0] (12)

24.8 [3.91 (23) 157.6 [25.6] (20)

aData are presented as means with [SEM] and (n)--number of animals (One animal of each sex from each litter). hA: Main effect of Ethanol; B: Main effect of Stress; AxB: Interaction; C: Nutritional effect; *p<0.05.

by ethanol-treated dams exhibited lower body weights on day 32 and lower brain weights at weaning. The stress treatment may have also affected postnatal care in the stressed dams as shown by decreased body weight at weaning in the prenatallyuntreated pups which had been fostered to stressed mothers. The fact that brain weight was significantly reduced on days 32 and 50 postconception due to prenatal ethanol exposure is consistent with the well-documented effects on brain growth found throughout the prenatal ethanol literature. The fact that prenatal stress did not reduce brain weight agrees with earlier findings reported in other laboratories (1,22). On the other hand, the effects on body weight in this study were less consistent, with significant reductions due to ethanol being found only at birth and at weaning, but not on day 32. The lack of an effect on body weight on day 32 in this strain of mouse has been found previously in our laboratory (18) suggesting that there may be a transient plateau in overall growth at this time which masks any differences in the rate of growth between groups. In contrast with our earlier findings (19), there was no main effect of prenatal stress in reducing the degree of neurobehavioral retardation produced by ethanol and pair feeding. This was not surprising since pair feeding in the present study did not, in fact, retard behavioral development in pups fostered to untreated dams. The reason for the discrepancy between the results of the stress treatment alone in this study and the stress effects reported elsewhere in the literature may be the fact that the current study controlled for postnatal maternal effects. The fact that prenatally stressed offspring were more active may account for the finding that they entered the shock com-

partment more quickly on the second day of shock avoidance conditioning. On the other hand, the fact that they did not enter more quickly on the first day suggests that avoidance learning had been differentially affected in these individuals. Whether or not such an effect can be interpreted as impaired avoidance conditioning is unclear since it would have to be established that there is an optimum time after which most animals will investigate the shock compartment regardless of their prior experience. Such an issue cannot be addressed by these findings. The present results, together with those reported in the earlier study (19), while consistently demonstrating harmful effects of prenatal ethanol exposure on morphological and behavioral development, provide little evidence to support the role of stressinduced physiological changes in contributing to such effects. A point worth mentioning is that this study, in c o m m o n with the others, used an acute model of stress. When considering factors which contribute to the neurobehavioral teratogenicity of ethanol, it is conceivable that a chronic model of stress may provide a more ecologically valid approach. ACKNOWLEDGEMENTS The authors thank Kathryn Blom and Morven Rennie of the Psychology Department and Chris Pelkrnan, Lisa Diflorio and Dawn McCutcheon of the Health Studies Department for their technical assistance. Doug Wahlsten of the Psychology Department provided helpful advice and Chris Young assisted with the statistical analysis. This research was conducted by G. R. Ward in partial fulfillment of the requirements for the Ph.D. degree in Psychology and was supported in part by a Natural Science and Engineering Council of Canada Grant A7617 awarded to P. Wainwright.

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