Prenatal ethanol and stress in mice: 1. Pup behavioral development and maternal physiology

Physiology & Behavior, Vol. 45, pp. 533-540. © Pergamon Press plc, 1989. Printed in the U.S.A.

0031-9384/89 $3.00 + .00

Prenatal Ethanol and Stress in Mice: 1. Pup Behavioral Development and Maternal Physiology G . R. W A R D * A N D P . E . W A I N W R I G H T t

l

*Department o f Psychology, tDepartment o f Health Studies, University o f Waterloo Waterloo, Ontario, N 2 L 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: 1. Pup behavioral development and maternal physiology. PHYSIOL BEHAV 45(3) 533-540, 1989.-On days 12 to 17 of pregnancy, B6D2FI mice were pair-fed liquid diets containing either 25% ethanol-derived calories or an isocaloric amount of maltose-dextrin. During this period, half the mice in each dietary condition also underwent two daily one-hour periods of restraint stress. A fifth group, given lab chow and water ad lib, was left undisturbed throughout gestation. Neither treatment affected offspring body weight on days 22 or 32 postconception, but undernutrition produced by the pair feeding procedure reduced day 32 body weight in all groups relative to the ad lib-fed group. Both prenatal ethanol and pair feeding led to delayed neurobehavioral development on day 32, while prenatal stress significantly reduced the degree of developmental delay caused by these factors. In a second study, restraint stress significantly reduced blood alcohol concentrations in pregnant dams on day 15 of gestation while elevating plasma corticosterone concentrations, and this elevation was consistent regardless of the dietary condition of the dam. The pair feeding procedure also produced corticosterone elevations but the effect of ethanol was not significant. These results suggest that prenatal stress in the presence of other physiological insults may act to counter the actions of those insults. Prenatal ethanol Restraint Mice

Prenatal stress

Neurobehavioral development

Corticosterone

Blood alcohol concentrations

teratogenic agents may act via stress-induced changes in maternal physiology (26). Furthermore, it is possible that these changes interact with the teratogen to exacerbate its biochemical effects. Therefore, we investigated the effects o f prenatal ethanol exposure combined with immobilization stress on behavioral development in mice. In this first paper we report two studies which demonstrate effects on reflex ontogeny and provide physiological validation of the experimental treatments in the pregnant dams. In the following paper, in which the role o f postnatal maternal factors is investigated, the findings include additional measures of brain and behavioral development.

R E S E A R C H E R S who study the effects of ethanol on experimental animals have reported a possible relationship between the effects of ethanol and those of stress (20). Acute ethanol administration increases circulating levels of both the adrenal steroid hormone, corticosterone (15,21), and the anterior pituitary hormone adrenocorticotropin ( A C T H ) (23). Although these increases may simply represent pharmacological responses to ethanol, they are often referred to as evidence that ethanol administration can have at least some o f the properties of a stressor. This latter notion has important implications for the study of prenatal ethanol effects since ethanol-induced corticosterone elevations have also been found during pregnancy (38). This is particularly interesting in light o f evidence that corticosterone crosses the placenta (42) and that some of the effects of prenatal ethanol exposure resemble those o f prenatal exposure to maternal stress. Studies on rodents report that both prenatal stress and prenatal ethanol exposure can produce increased frequency of cleft palate (2, 25, 37), increased susceptibility to audiogenic seizures (6,41), delayed sensorimotor development (4, 8, 10, 34), impaired performance on learning tasks (5,19), delayed female sexual maturation (7,22), increased lordotic behavior in males (13,36), and alterations in activity (9, 24, 40). Findings such as these have led some investigators to suggest that, in addition to their direct biochemical effects, certain

STUDY 1 In order to compare the effects of prenatal ethanol exposure and prenatal stress, and to examine possible interactive effects, it is necessary to measure a variable which consistently exhibits effects o f at least one o f these treatments. We have shown previously that B6D2F 2 hybrid mice exposed to ethanol from day 7 to 17 of gestation were significantly delayed in terms of neurobehavioral development (34). Animals were evaluated on tests o f reflex ontogeny, described in detail in an earlier paper (33), measuring such developmental landmarks as righting, cliff aversion, grasping, climbing, visual placing, eye opening, and auditory startle. The average score (x) was substituted into a

1Requests for reprints should be addressed to P. E. Wainwright.

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WARD AND WAINWRIGHT

quadratic regression equation of chronological age (y) on behavioral score (y=24.40 + l l.14x - 1.09x 2) to obtain the behavioral age in "B6D2F2 equivalent days postconception" (day 0=day of conception). This equation had been derived earlier by testing separate litters of B6D2F2 hybrid mice on each of gestation days 27 through 36 (31,32). Through the use of this scale, prenatal ethanol has been shown to retard development by 1.7 days relative to an untreated B6D2F2 control group (34). We therefore used this test battery to investigate the combined effects of ethanol consumption and physical restraint stress, which is reported to produce profound elevations in plasma corticosterone concentrations in mice (1,3), on days 12 to 17 of gestation.

TABLE 1 FORMULATION OF LIQUID DIETS*

o7o EDC 25 0

Sustacalt (ml)

95°7o EtoH (ml)

Maltose Dextrin (g)

Vitamin Mix$ (g)

Salt Mix§ (g)

73.5 73.5

4.7 0.0

0.0 6.4

0.30 0.30

0.25 0.25

*Tap water added to make final volume 100 ml with 1 kcal/ml. tMead Johnson and Company (chocolate flavored). $1CN Vitamin Diet Fortification Mixture No. 904654. §USP XIV.

METHOD

Subjects All B6D2F~ parental mice used in this study were obtained at four weeks of age from Charles River Breeding Laboratories, St. Constant, Quebec. Three to five animals were housed together in groups of the same sex with free access to laboratory chow (Master MLM rodent food, Maple Leaf Mills, Toronto) and tap water until mating commenced at 19 to 29 weeks of age. All maternal animals were primiparous. They were maintained under a reversed 12 hour dark:light cycle (dark 0800-2000 hr) in standard opaque plastic mouse cages (29× 18x13 cm) with Beta-Chip hardwood bedding and several sheets of toilet tissue for nesting material.

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) in the diet (25070 EDC or 0% EDC), with the 007o diet containing maltose-dextrin substituted isocalorically for ethanol. The formulation of the liquid diets is shown in Table 1. 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. Since pilot work showed that the mice undergoing the combined treatments consumed less diet than did mice receiving either treatment alone, each female was pair-fed during treatment to its weight matched partner in the group receiving both treatments. A fifth group was allowed free access to lab chow and water and was left undisturbed except for routine cage cleaning and weighing on days 12 and 17. This fifth group provided a control for the reliability of the behavioral scale and, when compared with the unstressed maltosedextrin group, allowed for the assessment of the effects of inadequate nutrition in the groups being pair-fed the liquid diet. The five experimental groups were designated as follows (n=No. of litters at birth: slight discrepancies between these figures and those reported in the tables reflect missing data due to random attrition or experimental oversight): Ethanol/Stress (E/S) (n= 10) Ethanol/No Stress (E/NS) (n=8) Maltose-Dextrin/Stress (MD/S) (n=9) Maltose-Dextrin/No stress (MD/NS) (n=9) Lab Chow (LC) (n=8)

Procedure One male was placed into each cage of females at the beginning of the dark cycle and removed seven hours later. At that time all females exhibiting vaginal plugs were weighed and housed individually. The day of conception was denoted as Day

0 postconception. On day l0 the dams were again weighed and on days l0 and 11 their lab chow and water were replaced by feeding them 0.625 kcal/g body weight of the 0°70 EDC liquid diet in order to accustom them to the diet while avoiding overfeeding due to its palatability. The daily amount was derived from pilot work on a similar population of pregnant animals. On day 12, the dams were assigned to groups on the basis of their body weight and fed the appropriate diet, the E/S group being allowed to feed ad lib while the other groups were pairfed on an individual basis to the E/S dams. 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. Animals receiving the liquid diet were weighed and fed fresh diet daily at the beginning of the dark cycle between 0800 hr and 0830 hr. The stressing procedure was adapted from Rosenzweig and Blaustein (25). A piece of wire mesh screen (20x20 cm) was folded over the mouse and the two halves were stapled together so as to restrict all voluntary movement other than slight twisting and turning. The restrained mouse was placed on four layers of toilet tissue and covered with a towel to help prevent possible heat loss due to immobility. All dams assigned to the stress condition were restrained for one hour commencing at 1030 hr and again seven hours later at 1730 hr from days 12 through 17. Unstressed groups (with the exception of the LC group) also had their food removed for each one-hour period. The pups were weighed on days 22 and 32 and, on day 32, two pups of each sex, selected randomly from each litter, underwent assessment of behavioral development by the test battery described above. All testing was done with the experimenter blind to the treatment condition of the animal. Because the mother had groomed the whiskers from the pups in some cases, one of the measures, vibrissae placing, was excluded from all scores in the final analysis.

Data Analysis The data were analysed using the general linear model (GLM) provided by the Statistical Analysis System (SAS) to do one way analysis of variance (ANOVA) with the experimental hypotheses, which are listed below, being addressed by preplanned linear contrasts. 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 Nutritional effect: LC vs. MD/NS

ETHANOL, STRESS AND BEHAVIORAL DEVELOPMENT

535

TABLE 2 EFFECTSON MATERNALAND PUP VARIABLESIN MICE OF ETHANOLEXPOSURECOMBINEDWITH RESTRAINTSTRESS ON DAYS 12-17 POSTCONCEPTION* Ethanol

Maternal Weight (g) Day 0

Day 12

Day 17

Litter Size

Pup Weight (g) Day 22

Day 32

Malt-Dex

Lab Chow

Stress

No Stress

Stress

No Stress

Control

25.00 [0.481 (10) 31.07 [0.76] (10) 36.98 [0.931 (10) 9.90 [0.66] (10)

24.95 [0.53] (8) 30.01 [0.52] (8) 35.59 [0.99] (8) 9.50 [0.50] (8)

24.87 [0.631 (9) 30.56 [0.75] (9) 36.12 [0.791 (9) 10.22 [0.46] (9)

25.17 [0.391 (9) 30.91 [0.84] (9) 38.69 [0.96] (9) 11.33 [0.41] (9)

26.53 [0.51] (6) 32.20 [0.67] (4) 44.57 [0.72] (7) 10.38 [0.18] (8)

2.04 [0.12] (9) 6.01 [0.261 (9)

1.82 [0.13] (8) 5.96 [0.331 (8)

2.06 [0.071 (9) 5.70 [0.131 (8)

2.04 [0.04] (9) 5.62 [0.13] (7)

2.28 [0.131 (5) 6.31 [0.251 (6)

Effects1"

AXB§ C~

A§

c¶

*Data are presented as means with [SEMI, and (n)=number of litters. Pup data are collapsed across sex. tA Main effect of Ethanol; B Main effect of Stress; AxB Interaction; C Nutritional effect. ~p
This approach is appropriate when the experimental hypotheses are presented a priori, requiring a specific set of comparisons to be made among the experimental groups (16). In the present case the first three comparisons comprise the orthogonal set addressing the main effects of ethanol and stress and their interaction, as would be done in a 2x2 factorial design, and the fourth evaluates nutritional effects due to pair-feeding the liquid diet. It should be noted, however, that the overall error term used to test each contrast is derived from all groups in the study, thereby making this a more powerful design than would separate analyses. The litter mean score was used as the unit of analysis, and alpha was set at p=0.05. RESULTS

Pup Variables As shown in Table 2, there were no significant differences among the groups in pup body weight on day 22 postconception, and on day 32 the only effect was that of nutrition, F(1,33)=3.62, p<0.05 (one-tailed), with the MD/NS pups being lighter than those in the LC group. Figure 1 shows main effects of both ethanol, F(1,33)=6.60, p<0.05, and stress, F(1,33)=4.45, p<0.05, on the behavioral development of the pups on day 32 postconception. Animals exposed to ethanol were 0.5 days behind those of the maltose-dextrin groups, whereas stressed animals were 0.5 days ahead of those of the nonstressed groups. There was no significant interaction but there was a delay of 0.6 days due to nutrition, F(1,33)=2.99, p<0.05 (one-tailed).

Maternal Variables Maternal data are also presented in Table 2. The data on days 0 and 12 confirm that all groups were matched on body weight. On day 17, there was a significant ethanol by stress interaction, F(1,38)=5.00, p<0.05, with the stressed dams being heavier only in the ethanol groups. There was a nutritional effect with the heaviest animals being those fed lab chow, F(1,38)=19.42, p<0.0001. The ethanol-fed dams had smaller litters by a magnitude of one pup, F(1,39)=4.92, p<0.05. STUDY 2 The results of the first study suggest that, while prenatal ethanol exposure retards behavioral development in mice, the effects of prenatal stress, in the presence of other physiological insults, may be beneficial. The second study was designed to provide physiological validation of the two experimental procedures. Previous research has shown that ethanol administration to pregnant rats increases the corticosterone response to stress (39) and also that undernutrition raises corticosterone levels in nonpregnant (29) and pregnant animals (27). Moreover, there is evidence that stress may affect blood alcohol concentrations (18). In this second study, we measured plasma corticosterone concentrations (PCC) and blood alcohol concentrations (BAC) on day 15 of gestation immediately following the one hour stress session, which in turn was initiated two hours after the onset of feeding at the beginning of the dark cycle.

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o

~

u

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o-

m

STRESS

l INOSTRE55

}g

ETHANOL

MALT-- DEX

LAB CHOW

PRENATALTREATMENT

FIG. 1. (Study 1). Effect of prenatal ethanol combined with restraint stress on the behavioral development of B6D2F2 mice on day 32 postconception. Values are group means _+ SEM. Ethanol p<0.001; Stress p<0.05; Nutrition p<0.05 (one-tailed).

METHOD

Subjects and Experimental Design The supplier and housing conditions of the parental mice were the same as in the first study. This was also true of the experimental design and analysis, with one exception. Because the BAC of both the stressed and unstressed dams receiving ethanol were measured at the end of the stress session, during which time no food was available, these measures would indicate levels resulting from two hours of ethanol consumption followed by one hour in which no ethanol was consumed. Therefore, an extra group of unstressed, ethanol-consuming dams was included to obtain BAC just prior to the stress session (i.e., a baseline measure following two hours of ethanol consumption during which this group was pair-fed in the same way as the experimental animals). The number of dams in each group was as follows: E/S, n=10; E/NS, n=9; E/NS (baseline), n=5; MD/S, n=10; MD/NS, n=9; LC, n=7.

Procedure The mating procedure, assignment to treatment groups, diet composition, and stress treatment were the same as in the first study. In mice, basal PCC rise during gestation, reaching their peak early in the third week (1), and the maximum stress-induced increase in PCC is reported to occur one hour after the initiation of physical restraint (3). In addition, feeding animals on a restricted diet at the beginning of the dark cycle has been shown to result in a circadian corticosterone rhythm similar to that of animals feeding ad lib (12). Therefore, in the present study, following the first stress session on day 15, the dam was killed by cervical dislocation, and 0.6 ml of blood was removed by cardiac puncture. Less than a minute elapsed between removal of the mouse from the experimental room and the cervical dislocation, and the blood was collected within the follow-

ing minute. The blood was centrifuged and the plasma was stored at - 8 0 ° C . Immediately following the collection of the blood, the fetuses were removed, blotted dry, and weighed to the nearest 0.1 mg on a Sartorius balance. Finally, in order to determine if differences in BAC were due to differences in ethanol consumption prior to the stress session, the amount of food that bad been consumed during that period was recorded.

Assays BAC were assessed using an adaptation of the ultraviolet NAD/NADH determination method outlined in the Sigma Technical Bulletin No. 332-UV, with all standards and reagents obtained from Sigma. All samples were analyzed concurrently and compared against standard solutions of 0, 0.05, 0.10, and 0.30% ethanol. All assays were run in duplicate. PCC were determined by radioimmunoassay using the ~251 Corticosterone Kit for rats and mice obtained from Radioassay Systems Laboratories, Inc., Carson, CA. Because of the exceptionally high PCC found in the pregnant mouse, the samples were diluted by a factor of 15. All standards and samples were run in triplicate. RESULTS

As is evident from Fig. 2, the ethanol feeding regimen resulted in BAC of 215 mg% in the baseline group (i.e., after two hours of consumption), whereas the values had dropped significantly during the next hour, F(2,21)=21.27, p<0.0001. The average amount of ethanol consumed during the first two hours did not differ significantly between the stressed and unstressed groups, but there was considerable within-group variability in consumption. Therefore, to control for this potential source of variability in BAC, a covariance analysis was carried out showing BAC, adjusted for differences in ethanol

ETHANOL, STRESS AND BEHAVIORAL DEVELOPMENT

537

LEGEND

1

i

I ETHANOL

200"

IooT

i

o

STRESS

NO

5TRESS

BASELINE

PRENATAL TREATMENT

FIG. 2. (Study 2). Blood alcohol concentration in stressed and unstressed pregnant B6D2Ft mice. Measures were taken on day 15 postconception at 1130 hr immediately following the 1 hour stress session. Baseline values were obtained from a group of similarly fed animals at 1030 hr immediately followingthe 2 hour period of ethanol consumption. Values are group means + SEM. Baseline vs. others p<0.001; Stress vs. No Stress p<0.05.

intake, to be significantly lower in the stressed dams, F(1,16)= 6.47, p<0.02. The plasma corticosterone concentrations are shown in Fig. 3. As expected, restraint stress produced a significant elevation in PCC, F(1,38)=78.92, p<0.0001, but there was no significant ethanol by stress interaction, F(1,38)=3.30, p<0.08. Although PCC were higher in the E/NS group compared with the MD/NS group, this was not statistically significant, F(1,38)=1.18, p<0.28. There was a nutritional effect, with the restriction in food intake in the pair-fed groups increasing PCC, F(1,38)=12.40, p<0.001, as well as reducing maternal body weight on day 15, F(1,40)=3.03, p<0.04 onetailed. As shown in Table 3, the ad lib fed group had smaller litters, F(1,40)=7.91, p<0.01. The only significant effect on fetal weight, shown in Fig. 4, was a significant reduction in weight due to the ethanol treatment, F(1,39)=11.85, p<0.001. GENERAL DISCUSSION In these experiments, prenatal restraint stress and prenatal ethanol consumption produced effects on behavioral development in B6D2F2 mice which were in opposite directions. Specifically, consumption of an ethanol diet capable of producing BAC's of 200 mg °7o led to developmental retardation in the offspring, as did the reduction in food intake due to the combined treatments. On the other hand, restraint stress, which produced dramatic elevations in plasma corticosterone concentrations in pregnant dams, led to an amelioration of the developmental retardation produced by both the ethanol and undernutrition. Other researchers have reported that various types of prenatal stressors accelerate development of specific postnatal phenomena in rats such as cliff avoidance, turning

upwards on an inclined plane, and swimming ability (10), righting (14), and the age of peak spontaneous motor activity (28). On the other hand, several studies have shown delayed behavioral and motor development following prenatal stress (4, 5, 11). It has been shown recently that common stressing procedures during pregnancy reduce food intake in the dams (17), and that many of the effects attributed to prenatal stress may be due, in fact, to stress-induced undernutrition (35). Therefore our results cannot be compared easily with those of the other studies due to the fact that the prenatally stressed offspring in this study exhibited accelerated development only when compared with their pair-fed controls. Had they been compared with the offspring of mothers which had been allowed unlimited access to food and water-the comparison that resembles most closely those made in the studies just c i t e d - t h e effect on neurobehaviorai development would not have been apparent. The PCC obtained here are consistent with those reported previously in pregnant mice after one hour of restraint stress (3). The increase in PCC in the stressed animals did not appear to be affected by the ethanol treatment and, although the PCC in the E/NS group were higher than those in the MD/NS group, this difference was not significant. The discrepancy between the findings in these stressed animals with the findings cited earlier (39) may be due to methodological differences between the studies. Another discrepancy from previous findings is that ethanol alone did not significantly elevate PCC over and above the levels found in the MD/NS group, contrary to earlier findings showing that consumption of a diet containing 25o70 EDC acted as a stressor in pregnant rats (38). This may be due to the pairfeeding procedure used in our study. Because we pair-fed all

538

WARD AND WAINWRIGHT

LEGEND

STRESS I

[ N O STRESS

87S"

7SO"

T T v)~

5oo.

375

MALT--DEX

ETHANOL

PRENATAL

LAB CHOW

TREATMENT

FIG. 3. (Study 2). Effect of prenatal ethanol combined with restraint stress on plasma corticosterone levels in pregnant B6D2Ft mice. Measures were taken on day 15 postconception at 1130 hr immediately following the 1 hour stress session. Values are group means _+ SEM. Stress p<0.001; Nutrition p<0.05.

TABLE 3 EFFECTS ON MATERNAL VARIABLES IN MICE OF E T H A N O L EXPOSURE COMBINED WITH RESTRAINT STRESS ON DAYS 12-15 POSTCONCEPTION*

Ethanol

Maternal Weight (g) Day 0

Day 12

Day 15

Litter Size

Malt-Dex

Lab Chow

Stress

No Stress

Stress

No Stress

Control

24.74 [0.401 (10) 29.03 [0.41] (10) 31.71 [0.47] (10) 9.60 [0.341 (10)

24.58 [0.431 (10) 28.89 [0.341 (10) 30.99 [0.53] (9) 9.78 [0.22] (9)

24.67 [0.511 (10) 28.50 [0.521 (10) 30.69 [0.751 (10) 9.50 [0.431 (10)

23.21 [0.75] (9) 28.11 [0.701 (9) 30.73 [0.92] (9) 9.44 [0.751 (9)

24.07 [0.571 (6) 28.27 [1.09] (7) 32.67 [1.01] (7) 7.14 [0.91] (7)

Effects~

c§¶ C§

*Data are presented as means with [SEM], and (n)=number of litters. I"A Main effect of Ethanol; B Main effect of Stress; A x B Interaction; C Nutritional effect. ~tp
treated groups to the E / S group, the reduction in food intake was matched for the combined effects of b o t h ethanol consumption a n d restraint stress r a t h e r t h a n for e t h a n o l c o n s u m p t i o n alone. Indeed, the pair-fed control g r o u p in our s t u d y - t h e

M D / N S g r o u p - - h a d significantly higher P C C t h a n did the LC control group. This increase due to undernutrition during gestation has been reported in at least one o t h e r study (27). The putative m e c h a n i s m o f the p r e n a t a l stress effect on

E T H A N O L , STRESS AND BEHAVIORAL D E V E L O P M E N T

539

LEGEND i~-:-:-:-:-:~ S T R E S S

[

INOSTRESS

¢~ .3"

.2'

.I

ETHANOL

MALT--DEX

PRENATAL

LAB C H O W

TREATMENT

FIG. 4. (Study 2). Effect of prenatal ethanol combined with restraint stress on fetal body weight on day 15 postconception. Values are group means _+ SEM. Ethanol p<0.001.

behavioral development is unclear. In the ethanol group, it could be related to the fact that the BAC in the stressed group were lower than those in the unstressed group. This could not have been due to differences in ethanol consumption between the groups since absolute daily intake, as well as the amount consumed within the first two hours, was the same in both groups. Restraint stress could act to increase the rate of metabolism of ethanol, and in this regard it is interesting to note that an early study reported that swimming stress lowered BAC in rats (18). At least one study (30) has reported that the administration of corticosterone is necessary for normal alcohol dehydrogenase (ADH) activity in the livers of adrenalectomised mice, suggesting that the levels of corticosterone may correlate with A D H activity in intact mice. Such a possibility warrants further study. In the maltose-dextrin grgups, the effect of prenatal stress on behavioral development cannot be explained from these results but could be due to differences in the pattern of food intake, or in the absorption of nutrients, or both. Besides reducing the amount of food consumed each day, restraint stress may also reduce the rate at which food is consumed. Thus, in the pair-feeding paradigm employed here, the stressed dams may prolong their daily period of feeding by consuming smaller quantities of food over a longer period of time. This altered pattern of feeding could conceivably result in more efficient uptake and utilization of nutrients by stressed dams. This is also an issue to be addressed by future research. These results confirm that prenatal ethanol exposure retards

behavioral development. With respect to prenatal stress, they support our previous findings (35) showing that, when nutritional intake is controlled between stressed and unstressed dams, the stress treatment does not appear to result in behavioral retardation. This suggests that stress-induced corticostetone elevations are not a major factor in the production of prenatal ethanol effects. In fact, in the present study, procedures which produce profound corticosterone elevations actually reduced some of the retardation in the ethanol-treated offspring. One point which should be made is that the treated pups in this study were reared by their own mothers, raising the possibility that some of the observed effects may have been mediated through postnatal maternal behavior and lactational performance. Therefore, it was considered important to replicate these results in a study which implemented fostering procedures to control postnatal maternal factors, and this is the subject of the next report. ACKNOWLEDGEMENTS The authors thank Kathryn Blom of the Psychology Department and Ian Fraser of the Kinesiology Department for their technical assistance, David Mills for advice regarding biochemical assays, and C. Young for statistical advice. This research represents a portion of the work conducted by G. R. Ward in partial fulfillment of the requirements for the Ph.D. degree in Psychology. It was supported in part by a Natural Science and Engineering Research Council of Canada Grant A7617 awarded to P. Wainwright with a contribution by Grant A4878 awarded to D. Wahlsten.

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