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Open-field and avoidance performance of rats as a function of prenatal ethanol treatment
4dd&tire Behacior,s. Vol. 4, pp. 311 to 322 © Pergamon Press Lld 1979. Prinled in Great Britain
0306-4603/79/1101-0311502.00/0
O P E N - F I E L D A N D AVOIDANCE P E R F O R M A N C E OF RATS AS A F U N C T I O N OF PRENATAL ETHANOL TREATMENT* WlLLIAM F. CAUL, GUY L. OSBORNE, KATHLEEN FERNANDEZ and GEORGE I. HENDERSON Departments of Psychologyand Pharmacology, Vanderbilt University, Nashville Abstrae~Pregnant rats were intubated during days 10-14 of gestation. Groups 6g, 4g, and 2g were intubated with one-half these amounts of ethanol twice each treatment day. Group PF animals were pair fed to group 6g animals and placebo intubated. Group C was not intubated. The treatments did not differentially affect the number or weight of pups delivered. Developmental measures taken prior to weaning were also not affected. Offspring behavior in an open field on days 63 and 64 was a function of maternal treatment with Group 4g showing increased ambulation and rearing. During Y-maze avoidance testing on days 65-69, Group 6g offspring made more avoidance responses than the other groups which were equivalent. Study 2 included offspring of dams given a 8g, 6g, 4g, or 2g treatment regimen. Treatment effects were observed in latency to move, rearing, and grooming in the open field. Although Group 8g and 6g dams were equivalent with regard to nutrition during gestation, Group 8g offspring made more avoidance responses and more correct discriminations in the Y maze than the other groups. Thus, in both studies, marked treatment-related behavioral effects were found after 60 days in rats that showed no developmental differences.
INTRODUCTION The reports of Lemoine et al. (1968), Ulleland (1972), and Jones & Smith (1973) have documented a pattern of growth retardation and morphological abnormalities in newborns associated with maternal alcoholism which has been termed the Fetal Alcohol Syndrome. Over 250 cases that vary in severity now have been reported (Clarren & Smith, 1978). In concluding their review, Clarren and Smith state that "The maternal abuse of ethanol during gestation produces a readily identifiable dysmorphic condition and appears to be the most frequent known teratogenic cause of mental deficiency in the western world" (Clarren & Smith, 1978, p, 1066). Children with the Fetal Alcohol Syndrome display a variety of disorders in physical development and behavior. Physical characteristics of the syndrome include growth deficiency, facial abnormalities, cardiac defects, and joint and limb anomalies. Experimental studies with animals that control for maternal nutrition and other factors relevant to prenatal development have yielded results consistent with these clinical observations (e.g., Tze & Lee, 1975; Randall et al., 1977; Kronik, 1976; Chernoff, 1977). Behavioral aspects of the Fetal Alcohol Syndrome as seen clinically include developmental delay, hyperactivity, and mental deficiency. Long-term intellectual impairment in children of alcoholic mothers (e.g., Streissguth, 1976; Streissguth et al., 1978) is the most serious behavioral problem associated with the Fetal Alcohol Syndrome. However, this effect cannot be unequivocably attributed to prenatal ethanol per se since socieconomic and other variables which define home environment cannot easily be eliminated as contributing factors (Rosett, 1974). It seems clear that an animal model is required to answer questions concerning altered learning ability. Few adequate studies exist, however, that address this issue. Some of the published studies are uninterpretable because of the methodological flaws, often related to appropriate nutritional controls (Randall, 1977), while others deal with behavior that cannot yield conclusions regarding learning per se. While altered sensitivity to audiogenic seizures (e.g. Yanai & Ginsburg, 1976) and altered movement in an open field (e.g., Branchey & Friedhoff, 1976) are *This investigation was supported in part by research grants from the Distilled Spirits Council of the United States, Inc., by NICHD No. 00973, and from The National Foundation--March of Dimes. AB4t4 A
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of interest, neither behavior is useful in direct evaluation of the effects of prenatal ethanol on associative (learning) and nonassociative (performance) processes. Appropriate assessment of this issue as related to prenatal ethanol treatment is thus badly needed. In attempting to elaborate possible behavioral effects of a known teratogen, it is not necessary to use doses that alter morphology. Although a positive correlation has been reported between the severity of dysmorphic features and degree of mental deficiency (Streissguth et al., 1978), there is no reason to believe that the absence of readily observable dysmorphic effects indicates the absence of subtle effects of prenatal ethanol that may influence behavior. Such functional teratogenic effects have been discussed in general by Wilson (e.g., 1973, 1975) and define the area of behavioral teratology (e.g., Werboff & Gottlieb, 1963; Barlow & Sullivan, 1975; Coyle et al., 1976). Research on behavioral teratogens may thus be of particular value in indicating the presence of insidious changes in anatomy and/or biochemistry which may otherwise go unnoticed (Coyle et al., 1976). This view suggests the need for research which uses a wide range of maternal ethanol dose in assessing postnatal development and behavior at maturity in offspring. The present study was therefore conducted to approach 3 goals. The first was to assess growth and developmental parameters in rats in offspring of dams given specified amounts of ethanol orally during days 10-14 of gestation. The second aim was to relate behavior in offspring of treated dams to the literature that suggests that increased ambulation results from prenatal ethanol exposure. Early studies by Vincent (1958) and Morra (1969) are suggestive at best in this regard, while alternative explanations exist for the findings of the more recent studies by Branchey & Friedhoff (1976) and by Bond & Di Giusto (1976, 1977). Branchey and Friedhoff provided ethanol in liquid diet from day 10 of gestation through the day of delivery while in the Bond and Di Giusto studies the liquid diet containing ethanol was available throughout the entire gestation period thus allowing the possibility that lingering effects of ethanol or its metabolites could affect the offspring postnatally via the mother's milk (Abel, 1974, 1975). Further the Bond and Di Giusto studies did not include appropriate pair-fed nutritional control treatments. The third aim of this study was to assess performance of offspring in a Y-maze avoidance task. This task requires that the animal learn to run to the lighted arm of a symmetrical Y maze in order to avoid or escape electric shock (cf. Osborne & Caul, 1975). Thus, the animal is required to learn "when" to run as well as "where" to run on the basis of a brightness discrimination. This advantage over the usual shuttlebox avoidance task, plus the availability of other measures of behavior in the Y maze, permit the analysis of treatment effects on behavior in terms of associative (learning) and non-associative (performance) factors. The multiple measures of behavior afforded by the Y-maze task have proven useful in separating associative and non-associative influences relative to the analysis of avoidance behavior in general (e.g., Barrett el al., 1973; Caul & Barrett, 1973), drug manipulations (e.g., Barrett et al., 1974), teratogenic effects of hypervitaminosis (Vorhees, 1974) and the effects of thiamine deficiency (Vorhees et al., 1975). MATERIALS
AND
METHODS
Nulliparous female Sprague-Dawley rats 101 days of age (260-280 g) were purchased from Holtzman Company, Madison, Wisconsin and housed individually for 17 days with free access to food and water. After this period, a male of the same stock was placed with each female. Males were removed when a semen plug was found during the 8 a.m. daily check of females and cage droppings. This day was designated gestation day 0. On gestation days 9-14, each female was fed powdered lab chow so that food consumption could be measured. Body weight was also recorded. Experimental treatment occurred on gestation days 10-14 at 6a.m. and again at 6 p.m. Groups 6g, 4g, and 2g received one-half these amounts of ethanol per kg of body weight p.o. at each treat-
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ment time. The ethanol solution consisted of ethanol (31.67~o v/v) in distilled water. G r o u p P F was pair fed to group 6g animals and was intubated with a sucrose solution. All p.o. treatments were of equal volume (12 ml/kg) and made to be equal in calories by the appropriate addition of sucrose. The procedure for G r o u p C was exactly as for the other 4 groups but did not include intubation. On gestation days 15-19 animals were weighed and food consumption assessed at 6 a.m. Immediately following this procedure on day 19, each female was placed in a cage measuring 48.3 x 26.7 x 21.5cm and provided bedding material of sugarcane waste. These preweaning cages were checked for the presence of pups 4 times" during the 6a.m. to 6 p.m. light cycle. Litters were assessed as to number, sex, and weight of pups between 1 and 4 hr after parturition. There were 4 litters in G r o u p 6g, 4 in G r o u p 4g, 3 in G r o u p 2g, 2 in G r o u p P F and 4 litters in G r o u p C. Although each dam in G r o u p 6g was originally paired with a P F dam, 1 P F animal failed to deliver and the other died as a result of intubation error. On alternate days 1 19 of the preweaning period each litter was assessed on a number of developmental parameters. After the mother was removed from the cage, each pup was examined to determine the presence of hair and teeth and the occurrence of eye openings. It was then placed in a small 25.5 × 25.5 cm enclosure divided into 25 equal squares by lines on the floor. During an observation period of 30sec head raising, squares entered, quality of walking, frequency of rearing against the wall, and grooming were recorded. The animal's weight was then recorded. After each pup was assessed on these measures, the mother was reintroduced into the preweaning cage. For the next 5 min the mother's behavior toward the pups was recorded in terms of retrievals, grooming, nursing, and contacts. Movement about the cage and nest manipulations were also recorded. This developmental assessment procedure took approximately 15 min per litter. On each of days 1 20 of the preweaning period spot checks of the litter and mother were made without disturbing the preweaning cage. Nine checks per day beginning at 8 a.m. were made at approximately 1 hr intervals. The number of pups nursing, pups with mother while not nursing, pups with other pups, and pups alone was noted during each of the 180 spot checks. Pups were weaned on day 21 and housed individually in standard laboratory cages. Food and water were provided ad libitum. On day 63 all animals were weighed and one-half of the males and one-half of the females of each litter were randomly selected to be tested in the open-field and Y-maze avoidance tasks. During behavioral testing, the identity of animals as to treatment group was unknown to the experimenter. On days 63 and 64 pups were tested individually in the open field which measured 80 x 80 cm and was divided into 16 equal squares by lines on the floor. Squares entered, rearing behavior, urination, and defecation were recorded during a 5 min observation period. Animals were given 20 trials of Y-maze avoidance training for 5 consecutive days starting on day 65. Three fully automated symmetrical Y mazes were used. The arms of the mazes were 27.9 × 17.8 x 19.5cm and were joined by a 17.8 x 17.8 x 17.8cm triangular choice area. Each maze was constructed of Plexiglas and had 0.3 cm diam grid floor bars spaced 1.9 cm apart. Opaque Plexiglas covered the top of the mazes. A 27-V d.c. lamp placed behind a translucent end wall in each arm served as the stimulus light. Footshock of 1.5-mA 60 Hz 205-V. a.c. was delivered to the grid floor through a scrambler. A fixed resistor (270 kfl) in series with the animal provided a relatively constant current. Responding was monitored with photocells placed 5 cm back from the entrance to each arm. The mazes were in a darkened room adjacent to the control room which housed standard relay equipment, timers, and digital counters used to program the behavioral contingencies and to accumulate response measures. A trial in the Y maze consisted of switching the stimulus light in random order to one of the dark arms. Entry into the lighted arm within 10sec successfully avoided shock. Failure to enter the lighted arm within the 10 sec period resulted in shock onset, after
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which escape responses were possible. Shock remained on in the previously safe arm and the incorrect arm as well as in the center triangular choice area until the animal entered the lighted safe arm. If, during the intertrial interval, the animal left the safe arm and broke the photobeam at the entrance of either of the dark arms, shock was initiated in the dark arms and in the center section and remained on until the animal returned to the safe arm. The following response measures were recorded during each Y-maze session: (a) avoidance--entry into the safe arm at any time during the 10sec C S - U S interval; (b) incorrect a v o i d a n c e s - - w h e n the initial response was an entry into the incorrect dark arm during the 10 sec CS-US interval; (c) correct discriminations-when the initial response (whether avoidance or escape) was an entry into the lighted safe arm; (d) incorrect e s c a p e s - - w h e n the initial response was an entry into the incorrect dark arm following shock onset; and (e) intertrial activity counts recorded as the animal m o v e d across the grid floor in the safe arm during the 30sec interval between trials. RESULTS
The effects of the treatments on the dams in terms of food consumption and body weight are seen in Fig. 1. During the 5 day treatment period the ethanol treatments produced decreased food consumption as a function of amount of ethanol intubated, F(16, 4 8 ) = 2.98, P < 0.01, and a corresponding decrease in body weight, F(16, 48) = 9.01, P < 0.001. The 6g and PF dams did not differ on either measure (F < 1 in each case). Following termination of the treatment, both food consumption, F(4, 48) = 22.17, P < 0.001, and body weight, F(4, 48) = 175.38, P < 0.001, increased during days 15-19. The rate of these changes was not dependent on treatment group.
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Table 1. Litter assessment, body weight, and sample size for behavioral testing as a function of maternal treatment Group Number of litters delivered Median length of gestation (days) Total viable pups Total dead pups at parturition Total viable male pups Total viable female pups Mean birth weight of males (g) Mean birth weight of females (g) Day 21 mean weight of males (g) Day 21 mean weight of females (g) Day 63 mean weight of males (g) Day 63 mean weight of females (g) Males used for behavioral testing (N) Females used for behavioral testing (N)
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Table 1 presents litter assessment data and mean birth weights for all pups. The mean weights for day 21 and day 63 were derived from only those animals eventually used in behavioral testing. Treatments did not differentially affect the number, sex ratio, weight or mortality rate of offspring. The only reliable factor in the analysis of birth weight was sex, i.e., males were heavier than females, F(1, 176) = 3.92, P < 0.05. This sex difference was not reliable at weaning on day 21, F(1, 7 6 ) < 1, but was highly reliable at day 63, F(1, 76) = 121, P < 0.001. While the difference in weight between males and females increased with age, F(1, 76) = 130.98, P < 0.001, there was no indication that the experimental treatments affected growth rate, F(4, 76) < 1. Analyses of the developmental data taken during the preweaning period with regard to weight gain, presence of hair, and presence of teeth showed orderly changes with age but no treatment effects. A reliable Treatment × Days effect F(36, 108) = 2.86, P < 0.001, was obtained, however, for eye openings. This result reflects the fact that G r o u p P F pups showed eye openings beginning on day 11 while this did not occur until day 13 for the ethanol and ad libitum control group pups. The development during the preweaning period of head raising, squares entered, quality of walking, frequency of rearing against the wall, and grooming was orderly as a function of age but, again, was not affected by the ethanol treatments. In the final phase of the developmental assessment procedure the mother's behavior toward the pups was recorded in terms of retrievals, grooming, nursing, and contacts with the pups. No treatment related differences in frequency of these behaviors were observed. Analyses of the data derived from the 180 unobtrusive spot checks during days 1-20 of the preweaning period also revealed no effects of the ethanol treatments on the behavior of the offspring or mother in the home cage. There was no evidence for group differences in percentage of pups nursing, pups with mother while not nursing, pups with other pups, and pups alone. Further, there was no suggestion of a Treatment x Days interaction with any measure. Figure 2 shows the mean ambulation score for both days of open-field testing. As is suggested by this figure, the analysis of these data showed a reliable Treatment x Days interaction, F(4, 76) = 6.96, P < 0.001. Subsequent analyses for each day showed that on day 1 females tended to enter more squares than did males, F(1, 76) = 3.82, P = 0.051, and that there was no evidence for an effect of Treatment, F(4, 76) = 1.07, P = 0.38. On day 2 females continued to ambulate more than males, F(1, 76) = 9.61, P < 0.01. In addition, there was also a significan t effect of maternal treatment, F(4, 76)--5.71, P < 0.001. F r o m Fig. 2 it can be seen that this group effect is attributable primarily to the marked increase in ambulation score for the G r o u p 4g animals in conjunction with the marked decrease in score for G r o u p C. The mean frequency of rearing during the 5 min open-field test shows a pattern of results parallel to those for ambulation scores in that females reared more than males
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b o t h on day 1, F(1, 76) = 23.77, P < 0.001, and on day 2, F(1, 76) = 35.32, P < 0.001, no Treatment effects occurred on day 1, F(4, 76) = 1.72, P = 0.15, and a reliable effect of T r e a t m e n t was found on day 2, F(4, 76) = 4.11, P < 0.001. As with the ambulation score, these results reflect an increase in rearing on day 2 for G r o u p 4g in conjunction with decreased rearing on day 2 for G r o u p s C and PF. Analyses of the other measures taken during open-field testing showed that males defecated, F(1, 76) = 6,58, P < 0.05, and urinated more than females, F(1, 76) = 18.40, P < 0.001. With neither measure was there evidence for a Treatment effect or a Treatment x Days interaction. The mean number of avoidance responses of all groups over the 5 days of Y-maze testing is shown on Fig. 3. The overall analysis of these data shows significant Days, F(4, 304) = 86.27, P < 0.001, and Treatment, F(4, 76) = 2.68, P < 0.05 effects. Further analyses indicated that this Treatment effect is attributable to the performance of G r o u p 6g. G r o u p 6g made more avoidance responses than its Pair Fed control group, F(I,
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29) = 5.16, P < 0.05, while Groups PF, C, 2g and 4g did not differ, F(3, 5 8 ) = 1.28, P = 0.263. In none of these analyses was the main effect of Sex or any interaction of Sex and other factors significant. The mean activity counts recorded in the safe arm during the intertrial interval for the groups during Y-maze sessions are seen in Fig. 4. As with the avoidance measure, there was a reliable Treatment effect, F(4, 76) = 3.63, P < 0.01. In addition, as suggested by Fig. 4, the Treatment x Days interaction was significant, F(16, 3 0 4 ) = 2.63, P < 0.001. An analysis of variance which included only the 6g and P F animals showed that activity levels were higher in group 6g, F(1, 29) = 10.05, P < 0.01, and that this difference was attenuated over days, F(4, 116) = 7.45, P < 0.001. Again there was no evidence for a difference in activity level in the Y maze between males and females. Analyses of the other measures taken in the Y maze of incorect avoidances, incorrect escapes, and total correct discriminations, indicated that there were no reliable Treatment or T r e a t m e n t × Days interactions.
DISCUSSION As seen in Fig. l, graded reductions in food consumption and body weight occurred as a function of ethanol dose during the treatment period. Behavioral observations of the females made 1 hr after intubation indicated that animals in the 6g group, i.e., 3g/kg per intubation, were slightly to moderately ataxic while animals in G r o u p 4g, i.e., 2g/kg per intubation were only occasionally slightly ataxic. In no case did these symptoms persist until the next intubation 12 hr later. When an 8g group was used as in study 2, the 4g/kg per intubation treatment produced moderate to severe ataxia and, occasionally, coma as observed 1 br after intubation. Again, as with the lower doses, these symptoms were absent 12 hr later. Pilot work using a 5g/kg per intubation dose at 12 hr intervals showed that this regimen is fatal to the animal. The assessment of litters at birth indicated no treatment-related effects on litter size or weight of pups. Abel (1978) reported reduced litter size and birth weight of pups after maternal treatment with 2g/kg per day ethanol throughout gestation. These effects, however were also seen in appropriat e pair fed control litters. The present study shows no effect on litters from dose as high as 6g/kg per day confined to gestation days 10-14. It should be noted, however, that differences in strain of rats used and number of intubations per day exist between the current study and that of Abel. Development with regard to weight gain, presence of hair, and presence of teeth showed orderly changes during the preweaning period consistent with data reported
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by Bolles & Woods (1964). No treatment effects were found on these measures or on the measures of pup behavior taken during the 21 days prior to weaning. The analysis of mother-infant interactions taken during developmental testing sessions as well as during the unobtrusive spot checks of maternal cages suggests the lack of treatment effects. The current study, however, did not attempt to make a detailed study of maternal behavior as might be done using the procedures of Rosenblatt & Lehrman (1963) or Grota & Ader (1969). In contrast to the litter assessment data and developmental data, there were marked behavioral changes as a function of maternal ethanol treatment observed in the openfield and Y-maze tests. The finding of increased ambulation and rearing on the second day of open-field testing especially in G r o u p 4g offspring relative to controls in the present study lends some support to the view that the results of Bond & Di Giusto (1976, 1977) and Branchey & Friedhoff (1976, 1977) are indeed attributable to the ethanol provided dams in the liquid diet during gestation. In the Y maze, the G r o u p 6g animals made more avoidance responses than the other groups. This finding must be viewed in relation to the other measures of behavior in the task. Not only did G r o u p 6g make more avoidance responses, but it was also clearly the most active during the intertrial interval in the safe arm, at least during the initial days of testing. Further, in terms of learning the correct discrimination that was required, these animals did not differ from those in the other treatment groups. Given this pattern of results it seems reasonable to suggest that the differences in avoidance responding were related to persistent treatment-produced differences in activity in this task rather than to altered learning ability. The application of this analysis to the passive-avoidance situation would lead to the prediction that offspring of dams given ethanol during gestation would perform more poorly than offspring of control dams. The results of Riley et al. (1979) support this prediction. While there is much support for this analysis of behavior in shock-avoidance situations (e.g., Caul & Barrett, 1973; Anisman & Waller, 1971), concern with this issue should not obscure the major finding of the present study. The results clearly show that maternal ethanol treatments during days 10-14 of gestation can produce long-term behavioral changes even though not affecting growth and development of the animal. STUDY 2
Study 2 had 4 purposes. The first was to replicate the 3 ethanol treatments of the first study with the addition of an 8g/kg per day group. The second was to assess maternal blood ethanol levels. The third was to replicate Study 1 with animals derived from litters of equal size. Although the treatments of Study 1 did not affect litter size, experimental control of this variable is desirable (e.g., G r o t a & Ader, 1969). The fourth purpose was to extend the dose range and assess open-field and Y-maze performance in animals not given the extensive handling and preweaning experience necessitated by the developmental testing procedures used in the first experiment.
MATERIALS AND METHODS Nulliparous females of the same strain of rat as used in Study 1 were mated at 120 days of age. Procedures were the same as previously described with 4 exceptions. (1) Four experimental treatments were used: 8g (N = 5), 6g (N = 6), 4g (N -= 6), and 2g (N = 5). (2) Blood ethanol levels were determined for all animals from samples taken 1 hr after each intubation on days 10, 12, and 14. Animals were lightly restrained while a 10/d sample of blood was extracted from a small cut made in the tail. Ethyl Alcohol StatPacks (Calbiochem) were used for determination of blood ethanol levels. (3) Food consumption and maternal body weights were monitored as before on gestation days 10-15 but not on days 16-19 as in the previous study. (4) Litters were assessed at delivery as in Study 1, culled to 4 males and 4 females when possible, and then left undisturbed until weaning at day "28.
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RESULTS Table 2 presents food c o n s u m p t i o n and b o d y weights for the d a m s during the treatm e n t period as well as b l o o d ethanol levels. As is a p p a r e n t from the means, over the treatment period, the dose level reliably affected the a m o u n t of food eaten, F(12, 72) = 13.16, P < 0.001, and maternal b o d y weight, F(12, 72) = 50.06, P < 0.001. Analyses c o m p a r i n g the 8g and 6g g r o u p s showed that these g r o u p s were equivalent in a m o u n t of food eaten, F(1, 36) < 1, and b o d y weight, F(I, 36) < 1, during the treatment period. B l o o d ethanol levels differed as a function of treatment, F(3, 1 8 ) = 24.30, P < 0.001, ranging f r o m the mean of 238.9 mg~o in the 8g g r o u p to the mean 81.4 mg~o in G r o u p 2g. Litter assessment data as well as offspring weights a n d sample size for behavioral testing are also seen on Table 2. As in the first experiment, the treatment did n o t differentially affect the length of gestation, the n u m b e r of pups delivered, or the male/ female ratio of offspring. The addition of the 8g g r o u p in this study p r o d u c e d a reliable treatment effect on offspring b o d y weight, F(3, 242) = 92.71, P < 0.001. The m e a n pup weight for G r o u p s 8g, 6g, 4g, and 2g was 5.66g, 6.59g, 7.14g, and 6.93 g, respectively. Even t h o u g h g r o u p s 8g and 6g did not differ in maternal food c o n s u m p t i o n or b o d y weight the offspring of 8g d a m s weighed less than the offspring of 6g dams, F(1, 122) = 75.49, P < 0.001. While males were heavier than females at birth, F(1, 242) = 17.82, P < 0.001, this sex difference did not interact with treatment effects. B o d y weight at weaning on day 28 and prior to testing on day 63 was analyzed with Age as a within-subject variable to assess possible differences in g r o w t h rate. The sex difference in weight was reliable, F(1, 152) = 118.81, P < 0.001, and increased in m a g n i t u d e with age, F(1, 150) = 100.74, P < 0.001. Neither the main effect of Treatment, F(3, 152) = 2.38, P = 0.07 nor the interaction o f T r e a t m e n t x Age, F(3, 150) = 1.67, P = 0.174, was reliable. Analysis of a m b u l a t i o n scores during the 2 days of open-field testing s h o w e d that there was no evidence for an effect of maternal treatment, F(3, 101) = 1.81, P = 0.149. T r e a t m e n t effects were seen, however, with the other measures of specific behaviors. L a t e n c y to enter a new square when first placed in the o p e n field was reliably affected by Treatment, F(3, 101) = 5.00, P < 0.01, with G r o u p 8g showing the shortest latency. T h e g r o u p means were 0.95 sec for G r o u p 8g, 1.36 sec for G r o u p 6g, 1.66 sec for G r o u p 4g, and 1.29 sec for G r o u p 2g. T r e a t m e n t effects were also found for rearing, F(3, 101) = 15.07, P < 0.001, and grooming, F(3, 91) = 2.77, P < 0.05. F r e q u e n c y of each Table 2. Maternal effects, litter assessment, body weight, and sample size for behavioral testing as a function of ethanol treatment Group Mean food eaten (g)/Mean body weight (g) Gestation day 11 12 13 14 15 Mean blood ethanol level (mg~o) Number of litters delivered Median length of gestation (days) Total viable pups Total dead pups at parturition Total viable male pups Total viable female pups Mean birth weights of males (g) Mean birth weights of females (g) Day 28 mean weight of males (g) Day 28 mean weight of females (g) Day 63 mean weight of males (g) Day 63 mean weight of females (g) Males used for behavioral testing (N) Females used for behavioral testing (N)
8g 24.3/330 1.8/296 0.4/282 0.0/269 0.1/258 238.9 5 22.00 59 0 36 23 5.77 5.55 87.13 78.07 282.44 195.14 9 11
6g 23.3/320 2.8/293 0.6/276 0.5/267 0.8/262 188.7 6 22.10 67 1 34 33 6.60 6.58 82.25 77.36 286.41 207.12 15 15
4g 24.1/314 9.9/302 14.4/308 17.3/313 15.9/315 109.6 6 22.00 60 2 34 26 7.36 6.92 86.28 80.1l 307.69 214.53 14 17
2g 23.2/314 14.6/312 16.5/314 17.5/318 21.2/326 81.4 5 21.88 64 0 31 33 7.16 6.70 89.42 82.90 286.90 230.80 14 14
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of these responses was greatest in offspring of G r o u p 8g dams. The means for rearing were: 8g = 27.30, 6g = 17.15, 4g = 12.47, 2g = 16.89. The means for grooming were: 8g = 0.86, 6g = 0.68, 4g = 0.56, 2g = 0.68. Consistent with Study 1, males crossed fewer squares, F(1, 1 0 1 ) = 5.26, P < 0.05, reared less often, F(1, 101) = 5.37, P < 0.05, defecated more, F(1, 101) = 6.38, P < 0.05, and urinated more, F(1, 101) = 7.96, P < 0.01, than females. These sex differences did not in any case interact with maternal treatment. The mean number of avoidance, responses as a function of maternal ethanol dose over the 5 days of Y-maze testing is seen on Fig. 5. Analysis of these data shows that there were reliable Treatment, F(3, 101) = 3.39, P < 0.05, Days, F(4, 404) = 121.10, P < 0.001, and Treatment x Days interaction, F(12, 404) = 2.00, P < 0.05, effects. The outstanding performance of 8g offspring provided the major contribution to this result. Although the groups were ordered as a function of maternal dose with the mean avoidances per day being 8.27 for G r o u p 8g, 5.89 for G r o u p 6g, 5.39 for G r o u p 4g, and 4.77 for G r o u p 2g, the differences over days a m o n g G r o u p s 6g, 4g, and 2g were not reliable, F(8, 332) <1. Again, with regard to correct discriminations, the 8g offspring stood out with a mean per day of 17.26 relative to means of 16.63 for group 6g, 16.33 for G r o u p 4g, and 16.17 for G r o u p 2g. The effects of both Treatment, F(3, 101)= 2.85, P < 0.05, and Days, F(4, 404) = 272.03, P < 0.001, were reliable. Given this difference in discrimination performance, a Treatment difference in incorrect escapes is expected, F(3, 101) = 2.95, P < 0.05, resulting from G r o u p 8g offspring making the fewest number of incorrect escapes. The analysis of incorrect avoidance responding yielded significant Days, F(4, 404) = 9.93, P < 0.001, and Treatment x Days, F(12, 404) = 2.06, P < 0.05, factors. The means, however, were not clearly ordered by maternal dose: 8g = 0.24, 6g = 0.17, 4g = 0.27, 2g = 0.17. While intertrial activity decreased over the 5 days of Y-maze testing, F(4, 391) = 32.08, P < 0.001, this decrease was not a function of treatment group. F(12, 391) < 1. DISCUSSION
In most measures taken in the open field and Y maze, the behavior of the 8g offspring stood out. During the 2 days of open tield testing they were faster to move initially, reared more and groomed more. In the Y maze, the 8g group made the most avoidance
Open-field and avoidance performance of rats
321
responses a n d also m a d e m o r e correct d i s c r i m i n a t i o n s t h a n the pups from the other t r e a t m e n t groups. T h e findings of this second study s u p p o r t a n d extend the results of the first experiment. T h e a p p a r e n t differences in the a b s o l u t e levels of the measures t a k e n in the o p e n field a n d Y maze between the 2 studies suggest a n influence of the extensive p r e w e a n i n g h a n d l i n g a n d experience given a n i m a l s of the first study d u r i n g d e v e l o p m e n t a l testing. Nevertheless, the results of b o t h studies clearly show that b e h a v i o r at days 63-69 was modified by p r e n a t a l e t h a n o l t r e a t m e n t . G i v e n that pair fed a n d a d l i b i t u m c o n t r o l s were n o t i n c l u d e d in S t u d y 2, it is i m p o r t a n t to n o t e that the 8g a n d 6g s o l u t i o n s i n t u b a t e d were equal in calories a n d that these 2 t r e a t m e n t s had e q u i v a l e n t effects o n m a t e r n a l food c o n s u m p t i o n a n d b o d y weight, Thus, the fact that the 8g a n d 6g d a m s were n u t r i t i o n a l l y the same, suggests that the dose-response curve seen in Fig. 5 s h o w i n g a v o i d a n c e b e h a v i o r is indicative of the effects of e t h a n o l p e r se. This evidence of l o n g - t e r m b e h a v i o r a l change following p r e n a t a l exposure to e t h a n o l indicates the i m p o r t a n c e of assessing f u n c t i o n a l t e r a t o g e n i c effects. G i v e n these findings, it is n o w i m p o r t a n t to elucidate the possible a n a t o m i c a l a n d / o r b i o c h e m i c a l m e c h a n i s m s u n d e r l y i n g such b e h a v i o r a l effects which occur w i t h o u t o b v i o u s physical teratogenic effects.
REFERENCES Abel, E. L. Alcohol ingestion in lactating rats: Effects on mothers and offspring. 1. Archives Internationales de Pharmacodynamie et de Therapie, 1974, 210, 121-127. Abel, E. L. Emotionality in offspring of rats fed alcohol while nursing. Journal of Studies on Alcohol, 1975, 36, 654-658. Abel, E. L. Effects of ethanol on pregnant rats and their offspring. Psychopharmacology, 1978, 57, 5 I I. Anisman, H. & Waller, T. G. Effects of inescapable shock upon subsequent one-way avoidance learning in two strains of rats. Psychonomic Science, 1971, 24, 101 102. Barlow, S. M. & Sullivan, F. M. Behavioral teratology. In C. L. Berry & D. E. Poswillo (Eds), Teratology: Trends and Applications. New York: Springer-Verlag, 1975. Barrett, R. J., Leith, N. J. & Ray, O. S. A behavioral and pharmacological analysis of variables mediating active-avoidance behavior in rats. Journal of Comparative and Physiological Psychology, 1973, 82, 489-500. Barrett, R. J., Leith, N. J. & Ray, O. S. An analysis of the facilitation of avoidance acquisition produced by d-Amphetamine and Scopolamine. Behavioral Biology, 1974, I I, 189-203. Bolles, R. C. & Woods, P. J. The ontogeny of behaviour in the albino rat. Animal Behaviour, 1964, 12, 427M.41. Bond, N. W. & Di Giusto, E. L. Effects of prenatal alcohol consumption on open-field behaviour and alcohol preference in rats. Psychopharmqcologia (Berl.), 1976, 46, 163-165. Bond, N. W. & Di Giusto, E. L. Prenatal alcohol consumption and open-field behaviour in rats: Effects of age at time of testing. Psychopharmacology, 1977, 52, 311-312. Branchey, L. & Friedhoff, A. J. Biochemical and behavioral changes in rats exposed to ethanol in utero. Annals of the New York Academy of Sciences, 1976, 273, 328-330. Caul, W. F. & Barrett, R. J. Shuttle-box versus Y-maze avoidance: Value of multiple response measures in interpreting active-avoidance performance of rats. Journal of Comparative and Physiological Psychology, 1973, 84, 572-578. Chernoff, G. F. The fetal alcohol syndrome in mice: An animal model. Teratology, 1977, 15, 223-230. Clarren, S. K. & Smith, D. W. The fetal alcohol syndrome. New England Journal of Medicine, 1978, 298, 1063-1067. Coyle, I., Wayner, M. J. & Singer, G. Behavioral teratogenesis: A critical evaluation. Pharmacology, Biochemistry and Behavior, 1976, 4, 191-200. Grota, L. J. & Ader, R. Continuous recording of maternal behaviour in Rattus norvegicus. Animal Behaviour, 1969, 17, 722-729. Jones, K. L. & Smith, D. W. Recognition of the fetal alcohol syndrome in early infancy. Lancet, 1973, II, 999-1001. Kronick, J. B. Teratogenic effects of ethyl alcohol administered to pregnant mice. American Journal of Obstetrics and Gynecology, 1976, 124, 6764580. Lemoine, P., Harousseau, H., Borteyru, J.-P. & Menuet, J.-C. Les enfants de parents alcooliques: anomalies observ6es ~. propos de 127 cas. Quest Medical, 1968, 25, 47~482. Morra, M. Ethanol and maternal stress on rat offspring behaviors. Journal of Genetic Psychology, 1969, i14, 77-83. Osborne, G. L. & Caul, W. F. Y-maze avoidance performance and activity change as a function of CS quality in rats. Behavioral Biology, 1975, 15, 183-192. Randall, C. L. Teratogenic effect of in utero ethanol exposure. In K. Blum (Ed.), Alcohol and Opiates: Neuroehemical and Behavioral Mechanisms. New York: Academic Press, 1977. Randall, C. L., Taylor, W. J. & Walker, D. W. Ethanol-induced malformations in mice. Alcoholism Clinieal and Experimental Researeh, 1977, I, 219 223.
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Riley, E. P., Lochry, E. & Shapiro, N. Lack of response inhibition in rats prenatally exposed to alcohol. Psychopharmaeology, 1979, In press. Rosenblatt, J. S. & Lehrman, D. S. Material behavior of the laboratory rat. In H. S. Reingold (Ed.), Maternal Behavior of Mammals. New York: Wiley, 1963. Rosett, H. L. Maternal alcoholism and intellectual development of offspring. Lancet, 1974, 2, 218. Streissguth, A. P. Psychologic handicaps in children with the fetal alcohol syndrome. Annals of the New York Academy of Science, 1976, 273, 14(~145. Streissguth, A. P., Herman, C. S. & Smith, D. W. Intelligence, behavior and dysmorphogenesis in the fetal alcohol syndrome: a report on 20 patients. Journal of Pediatrics, 1978, 92, 363 367. Tze, W. J. & Lee, M. Adverse effects of maternal alcohol consumption on pregnancy and foetal growth in rats. Nature, 1975, 257, 479-480. Ulleland, C. W. The offspring of alcoholic mothers. Annals ~?["the New York Academy o.1 Sciences, 1972, 197, 167-169. Vincent, N. M. The effects of prenatal alcoholism upon motivation, emotionality and learning. American Psychologist, 1958, 13, 401. Vorhees, C. V. Some behavioral effects of maternal hypervitaminosis A in rats. Teratology, 1974, 10, 269-273. Vorhees, C. V., Barrett, R. J. & Schenker, S. Increased muricide and decreased avoidance and discrimination learning in thiamine deficient rats. Life Sciences, 1975, 16, 1187-1200. Werboff, J. & Gottlieb, J. S. Drugs in pregnancy: Behavioral teratology, Obstetrics and Gynecology Survey, 1963, 18, 420-423. Wilson, J. G. Environment and Birth Defects. New York: Academic Press, 1973. Wilson, J. G. Critique of current methods for teratogenicity testing in animals and suggestions for their improvement. In T. H. Shepard, J. R. Miller & M. Marois (Eds), Methods for Detection of Environmental Agents That Produce Congenital Dejects. New York: Elsevier, 1975. Yanai, J. & Ginsburg, B. E. Audiogenic seizures in mice whose parents drank alcohol. Journal of Studies on Alcohol, 1976, 37, 1564--1571.
0306-4603/79/1101-0311502.00/0
O P E N - F I E L D A N D AVOIDANCE P E R F O R M A N C E OF RATS AS A F U N C T I O N OF PRENATAL ETHANOL TREATMENT* WlLLIAM F. CAUL, GUY L. OSBORNE, KATHLEEN FERNANDEZ and GEORGE I. HENDERSON Departments of Psychologyand Pharmacology, Vanderbilt University, Nashville Abstrae~Pregnant rats were intubated during days 10-14 of gestation. Groups 6g, 4g, and 2g were intubated with one-half these amounts of ethanol twice each treatment day. Group PF animals were pair fed to group 6g animals and placebo intubated. Group C was not intubated. The treatments did not differentially affect the number or weight of pups delivered. Developmental measures taken prior to weaning were also not affected. Offspring behavior in an open field on days 63 and 64 was a function of maternal treatment with Group 4g showing increased ambulation and rearing. During Y-maze avoidance testing on days 65-69, Group 6g offspring made more avoidance responses than the other groups which were equivalent. Study 2 included offspring of dams given a 8g, 6g, 4g, or 2g treatment regimen. Treatment effects were observed in latency to move, rearing, and grooming in the open field. Although Group 8g and 6g dams were equivalent with regard to nutrition during gestation, Group 8g offspring made more avoidance responses and more correct discriminations in the Y maze than the other groups. Thus, in both studies, marked treatment-related behavioral effects were found after 60 days in rats that showed no developmental differences.
INTRODUCTION The reports of Lemoine et al. (1968), Ulleland (1972), and Jones & Smith (1973) have documented a pattern of growth retardation and morphological abnormalities in newborns associated with maternal alcoholism which has been termed the Fetal Alcohol Syndrome. Over 250 cases that vary in severity now have been reported (Clarren & Smith, 1978). In concluding their review, Clarren and Smith state that "The maternal abuse of ethanol during gestation produces a readily identifiable dysmorphic condition and appears to be the most frequent known teratogenic cause of mental deficiency in the western world" (Clarren & Smith, 1978, p, 1066). Children with the Fetal Alcohol Syndrome display a variety of disorders in physical development and behavior. Physical characteristics of the syndrome include growth deficiency, facial abnormalities, cardiac defects, and joint and limb anomalies. Experimental studies with animals that control for maternal nutrition and other factors relevant to prenatal development have yielded results consistent with these clinical observations (e.g., Tze & Lee, 1975; Randall et al., 1977; Kronik, 1976; Chernoff, 1977). Behavioral aspects of the Fetal Alcohol Syndrome as seen clinically include developmental delay, hyperactivity, and mental deficiency. Long-term intellectual impairment in children of alcoholic mothers (e.g., Streissguth, 1976; Streissguth et al., 1978) is the most serious behavioral problem associated with the Fetal Alcohol Syndrome. However, this effect cannot be unequivocably attributed to prenatal ethanol per se since socieconomic and other variables which define home environment cannot easily be eliminated as contributing factors (Rosett, 1974). It seems clear that an animal model is required to answer questions concerning altered learning ability. Few adequate studies exist, however, that address this issue. Some of the published studies are uninterpretable because of the methodological flaws, often related to appropriate nutritional controls (Randall, 1977), while others deal with behavior that cannot yield conclusions regarding learning per se. While altered sensitivity to audiogenic seizures (e.g. Yanai & Ginsburg, 1976) and altered movement in an open field (e.g., Branchey & Friedhoff, 1976) are *This investigation was supported in part by research grants from the Distilled Spirits Council of the United States, Inc., by NICHD No. 00973, and from The National Foundation--March of Dimes. AB4t4 A
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of interest, neither behavior is useful in direct evaluation of the effects of prenatal ethanol on associative (learning) and nonassociative (performance) processes. Appropriate assessment of this issue as related to prenatal ethanol treatment is thus badly needed. In attempting to elaborate possible behavioral effects of a known teratogen, it is not necessary to use doses that alter morphology. Although a positive correlation has been reported between the severity of dysmorphic features and degree of mental deficiency (Streissguth et al., 1978), there is no reason to believe that the absence of readily observable dysmorphic effects indicates the absence of subtle effects of prenatal ethanol that may influence behavior. Such functional teratogenic effects have been discussed in general by Wilson (e.g., 1973, 1975) and define the area of behavioral teratology (e.g., Werboff & Gottlieb, 1963; Barlow & Sullivan, 1975; Coyle et al., 1976). Research on behavioral teratogens may thus be of particular value in indicating the presence of insidious changes in anatomy and/or biochemistry which may otherwise go unnoticed (Coyle et al., 1976). This view suggests the need for research which uses a wide range of maternal ethanol dose in assessing postnatal development and behavior at maturity in offspring. The present study was therefore conducted to approach 3 goals. The first was to assess growth and developmental parameters in rats in offspring of dams given specified amounts of ethanol orally during days 10-14 of gestation. The second aim was to relate behavior in offspring of treated dams to the literature that suggests that increased ambulation results from prenatal ethanol exposure. Early studies by Vincent (1958) and Morra (1969) are suggestive at best in this regard, while alternative explanations exist for the findings of the more recent studies by Branchey & Friedhoff (1976) and by Bond & Di Giusto (1976, 1977). Branchey and Friedhoff provided ethanol in liquid diet from day 10 of gestation through the day of delivery while in the Bond and Di Giusto studies the liquid diet containing ethanol was available throughout the entire gestation period thus allowing the possibility that lingering effects of ethanol or its metabolites could affect the offspring postnatally via the mother's milk (Abel, 1974, 1975). Further the Bond and Di Giusto studies did not include appropriate pair-fed nutritional control treatments. The third aim of this study was to assess performance of offspring in a Y-maze avoidance task. This task requires that the animal learn to run to the lighted arm of a symmetrical Y maze in order to avoid or escape electric shock (cf. Osborne & Caul, 1975). Thus, the animal is required to learn "when" to run as well as "where" to run on the basis of a brightness discrimination. This advantage over the usual shuttlebox avoidance task, plus the availability of other measures of behavior in the Y maze, permit the analysis of treatment effects on behavior in terms of associative (learning) and non-associative (performance) factors. The multiple measures of behavior afforded by the Y-maze task have proven useful in separating associative and non-associative influences relative to the analysis of avoidance behavior in general (e.g., Barrett el al., 1973; Caul & Barrett, 1973), drug manipulations (e.g., Barrett et al., 1974), teratogenic effects of hypervitaminosis (Vorhees, 1974) and the effects of thiamine deficiency (Vorhees et al., 1975). MATERIALS
AND
METHODS
Nulliparous female Sprague-Dawley rats 101 days of age (260-280 g) were purchased from Holtzman Company, Madison, Wisconsin and housed individually for 17 days with free access to food and water. After this period, a male of the same stock was placed with each female. Males were removed when a semen plug was found during the 8 a.m. daily check of females and cage droppings. This day was designated gestation day 0. On gestation days 9-14, each female was fed powdered lab chow so that food consumption could be measured. Body weight was also recorded. Experimental treatment occurred on gestation days 10-14 at 6a.m. and again at 6 p.m. Groups 6g, 4g, and 2g received one-half these amounts of ethanol per kg of body weight p.o. at each treat-
Open-field and avoidance performance of rats
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ment time. The ethanol solution consisted of ethanol (31.67~o v/v) in distilled water. G r o u p P F was pair fed to group 6g animals and was intubated with a sucrose solution. All p.o. treatments were of equal volume (12 ml/kg) and made to be equal in calories by the appropriate addition of sucrose. The procedure for G r o u p C was exactly as for the other 4 groups but did not include intubation. On gestation days 15-19 animals were weighed and food consumption assessed at 6 a.m. Immediately following this procedure on day 19, each female was placed in a cage measuring 48.3 x 26.7 x 21.5cm and provided bedding material of sugarcane waste. These preweaning cages were checked for the presence of pups 4 times" during the 6a.m. to 6 p.m. light cycle. Litters were assessed as to number, sex, and weight of pups between 1 and 4 hr after parturition. There were 4 litters in G r o u p 6g, 4 in G r o u p 4g, 3 in G r o u p 2g, 2 in G r o u p P F and 4 litters in G r o u p C. Although each dam in G r o u p 6g was originally paired with a P F dam, 1 P F animal failed to deliver and the other died as a result of intubation error. On alternate days 1 19 of the preweaning period each litter was assessed on a number of developmental parameters. After the mother was removed from the cage, each pup was examined to determine the presence of hair and teeth and the occurrence of eye openings. It was then placed in a small 25.5 × 25.5 cm enclosure divided into 25 equal squares by lines on the floor. During an observation period of 30sec head raising, squares entered, quality of walking, frequency of rearing against the wall, and grooming were recorded. The animal's weight was then recorded. After each pup was assessed on these measures, the mother was reintroduced into the preweaning cage. For the next 5 min the mother's behavior toward the pups was recorded in terms of retrievals, grooming, nursing, and contacts. Movement about the cage and nest manipulations were also recorded. This developmental assessment procedure took approximately 15 min per litter. On each of days 1 20 of the preweaning period spot checks of the litter and mother were made without disturbing the preweaning cage. Nine checks per day beginning at 8 a.m. were made at approximately 1 hr intervals. The number of pups nursing, pups with mother while not nursing, pups with other pups, and pups alone was noted during each of the 180 spot checks. Pups were weaned on day 21 and housed individually in standard laboratory cages. Food and water were provided ad libitum. On day 63 all animals were weighed and one-half of the males and one-half of the females of each litter were randomly selected to be tested in the open-field and Y-maze avoidance tasks. During behavioral testing, the identity of animals as to treatment group was unknown to the experimenter. On days 63 and 64 pups were tested individually in the open field which measured 80 x 80 cm and was divided into 16 equal squares by lines on the floor. Squares entered, rearing behavior, urination, and defecation were recorded during a 5 min observation period. Animals were given 20 trials of Y-maze avoidance training for 5 consecutive days starting on day 65. Three fully automated symmetrical Y mazes were used. The arms of the mazes were 27.9 × 17.8 x 19.5cm and were joined by a 17.8 x 17.8 x 17.8cm triangular choice area. Each maze was constructed of Plexiglas and had 0.3 cm diam grid floor bars spaced 1.9 cm apart. Opaque Plexiglas covered the top of the mazes. A 27-V d.c. lamp placed behind a translucent end wall in each arm served as the stimulus light. Footshock of 1.5-mA 60 Hz 205-V. a.c. was delivered to the grid floor through a scrambler. A fixed resistor (270 kfl) in series with the animal provided a relatively constant current. Responding was monitored with photocells placed 5 cm back from the entrance to each arm. The mazes were in a darkened room adjacent to the control room which housed standard relay equipment, timers, and digital counters used to program the behavioral contingencies and to accumulate response measures. A trial in the Y maze consisted of switching the stimulus light in random order to one of the dark arms. Entry into the lighted arm within 10sec successfully avoided shock. Failure to enter the lighted arm within the 10 sec period resulted in shock onset, after
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which escape responses were possible. Shock remained on in the previously safe arm and the incorrect arm as well as in the center triangular choice area until the animal entered the lighted safe arm. If, during the intertrial interval, the animal left the safe arm and broke the photobeam at the entrance of either of the dark arms, shock was initiated in the dark arms and in the center section and remained on until the animal returned to the safe arm. The following response measures were recorded during each Y-maze session: (a) avoidance--entry into the safe arm at any time during the 10sec C S - U S interval; (b) incorrect a v o i d a n c e s - - w h e n the initial response was an entry into the incorrect dark arm during the 10 sec CS-US interval; (c) correct discriminations-when the initial response (whether avoidance or escape) was an entry into the lighted safe arm; (d) incorrect e s c a p e s - - w h e n the initial response was an entry into the incorrect dark arm following shock onset; and (e) intertrial activity counts recorded as the animal m o v e d across the grid floor in the safe arm during the 30sec interval between trials. RESULTS
The effects of the treatments on the dams in terms of food consumption and body weight are seen in Fig. 1. During the 5 day treatment period the ethanol treatments produced decreased food consumption as a function of amount of ethanol intubated, F(16, 4 8 ) = 2.98, P < 0.01, and a corresponding decrease in body weight, F(16, 48) = 9.01, P < 0.001. The 6g and PF dams did not differ on either measure (F < 1 in each case). Following termination of the treatment, both food consumption, F(4, 48) = 22.17, P < 0.001, and body weight, F(4, 48) = 175.38, P < 0.001, increased during days 15-19. The rate of these changes was not dependent on treatment group.
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Open-field and avoidance performance of rats
315
Table 1. Litter assessment, body weight, and sample size for behavioral testing as a function of maternal treatment Group Number of litters delivered Median length of gestation (days) Total viable pups Total dead pups at parturition Total viable male pups Total viable female pups Mean birth weight of males (g) Mean birth weight of females (g) Day 21 mean weight of males (g) Day 21 mean weight of females (g) Day 63 mean weight of males (g) Day 63 mean weight of females (g) Males used for behavioral testing (N) Females used for behavioral testing (N)
6g 4 22.00 39 3 21 18 7.00 6.46 54.62 55.53 328.93 237.22 11 9
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Table 1 presents litter assessment data and mean birth weights for all pups. The mean weights for day 21 and day 63 were derived from only those animals eventually used in behavioral testing. Treatments did not differentially affect the number, sex ratio, weight or mortality rate of offspring. The only reliable factor in the analysis of birth weight was sex, i.e., males were heavier than females, F(1, 176) = 3.92, P < 0.05. This sex difference was not reliable at weaning on day 21, F(1, 7 6 ) < 1, but was highly reliable at day 63, F(1, 76) = 121, P < 0.001. While the difference in weight between males and females increased with age, F(1, 76) = 130.98, P < 0.001, there was no indication that the experimental treatments affected growth rate, F(4, 76) < 1. Analyses of the developmental data taken during the preweaning period with regard to weight gain, presence of hair, and presence of teeth showed orderly changes with age but no treatment effects. A reliable Treatment × Days effect F(36, 108) = 2.86, P < 0.001, was obtained, however, for eye openings. This result reflects the fact that G r o u p P F pups showed eye openings beginning on day 11 while this did not occur until day 13 for the ethanol and ad libitum control group pups. The development during the preweaning period of head raising, squares entered, quality of walking, frequency of rearing against the wall, and grooming was orderly as a function of age but, again, was not affected by the ethanol treatments. In the final phase of the developmental assessment procedure the mother's behavior toward the pups was recorded in terms of retrievals, grooming, nursing, and contacts with the pups. No treatment related differences in frequency of these behaviors were observed. Analyses of the data derived from the 180 unobtrusive spot checks during days 1-20 of the preweaning period also revealed no effects of the ethanol treatments on the behavior of the offspring or mother in the home cage. There was no evidence for group differences in percentage of pups nursing, pups with mother while not nursing, pups with other pups, and pups alone. Further, there was no suggestion of a Treatment x Days interaction with any measure. Figure 2 shows the mean ambulation score for both days of open-field testing. As is suggested by this figure, the analysis of these data showed a reliable Treatment x Days interaction, F(4, 76) = 6.96, P < 0.001. Subsequent analyses for each day showed that on day 1 females tended to enter more squares than did males, F(1, 76) = 3.82, P = 0.051, and that there was no evidence for an effect of Treatment, F(4, 76) = 1.07, P = 0.38. On day 2 females continued to ambulate more than males, F(1, 76) = 9.61, P < 0.01. In addition, there was also a significan t effect of maternal treatment, F(4, 76)--5.71, P < 0.001. F r o m Fig. 2 it can be seen that this group effect is attributable primarily to the marked increase in ambulation score for the G r o u p 4g animals in conjunction with the marked decrease in score for G r o u p C. The mean frequency of rearing during the 5 min open-field test shows a pattern of results parallel to those for ambulation scores in that females reared more than males
316
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b o t h on day 1, F(1, 76) = 23.77, P < 0.001, and on day 2, F(1, 76) = 35.32, P < 0.001, no Treatment effects occurred on day 1, F(4, 76) = 1.72, P = 0.15, and a reliable effect of T r e a t m e n t was found on day 2, F(4, 76) = 4.11, P < 0.001. As with the ambulation score, these results reflect an increase in rearing on day 2 for G r o u p 4g in conjunction with decreased rearing on day 2 for G r o u p s C and PF. Analyses of the other measures taken during open-field testing showed that males defecated, F(1, 76) = 6,58, P < 0.05, and urinated more than females, F(1, 76) = 18.40, P < 0.001. With neither measure was there evidence for a Treatment effect or a Treatment x Days interaction. The mean number of avoidance responses of all groups over the 5 days of Y-maze testing is shown on Fig. 3. The overall analysis of these data shows significant Days, F(4, 304) = 86.27, P < 0.001, and Treatment, F(4, 76) = 2.68, P < 0.05 effects. Further analyses indicated that this Treatment effect is attributable to the performance of G r o u p 6g. G r o u p 6g made more avoidance responses than its Pair Fed control group, F(I,
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29) = 5.16, P < 0.05, while Groups PF, C, 2g and 4g did not differ, F(3, 5 8 ) = 1.28, P = 0.263. In none of these analyses was the main effect of Sex or any interaction of Sex and other factors significant. The mean activity counts recorded in the safe arm during the intertrial interval for the groups during Y-maze sessions are seen in Fig. 4. As with the avoidance measure, there was a reliable Treatment effect, F(4, 76) = 3.63, P < 0.01. In addition, as suggested by Fig. 4, the Treatment x Days interaction was significant, F(16, 3 0 4 ) = 2.63, P < 0.001. An analysis of variance which included only the 6g and P F animals showed that activity levels were higher in group 6g, F(1, 29) = 10.05, P < 0.01, and that this difference was attenuated over days, F(4, 116) = 7.45, P < 0.001. Again there was no evidence for a difference in activity level in the Y maze between males and females. Analyses of the other measures taken in the Y maze of incorect avoidances, incorrect escapes, and total correct discriminations, indicated that there were no reliable Treatment or T r e a t m e n t × Days interactions.
DISCUSSION As seen in Fig. l, graded reductions in food consumption and body weight occurred as a function of ethanol dose during the treatment period. Behavioral observations of the females made 1 hr after intubation indicated that animals in the 6g group, i.e., 3g/kg per intubation, were slightly to moderately ataxic while animals in G r o u p 4g, i.e., 2g/kg per intubation were only occasionally slightly ataxic. In no case did these symptoms persist until the next intubation 12 hr later. When an 8g group was used as in study 2, the 4g/kg per intubation treatment produced moderate to severe ataxia and, occasionally, coma as observed 1 br after intubation. Again, as with the lower doses, these symptoms were absent 12 hr later. Pilot work using a 5g/kg per intubation dose at 12 hr intervals showed that this regimen is fatal to the animal. The assessment of litters at birth indicated no treatment-related effects on litter size or weight of pups. Abel (1978) reported reduced litter size and birth weight of pups after maternal treatment with 2g/kg per day ethanol throughout gestation. These effects, however were also seen in appropriat e pair fed control litters. The present study shows no effect on litters from dose as high as 6g/kg per day confined to gestation days 10-14. It should be noted, however, that differences in strain of rats used and number of intubations per day exist between the current study and that of Abel. Development with regard to weight gain, presence of hair, and presence of teeth showed orderly changes during the preweaning period consistent with data reported
318
WILLIAM F. CAUL et al.
by Bolles & Woods (1964). No treatment effects were found on these measures or on the measures of pup behavior taken during the 21 days prior to weaning. The analysis of mother-infant interactions taken during developmental testing sessions as well as during the unobtrusive spot checks of maternal cages suggests the lack of treatment effects. The current study, however, did not attempt to make a detailed study of maternal behavior as might be done using the procedures of Rosenblatt & Lehrman (1963) or Grota & Ader (1969). In contrast to the litter assessment data and developmental data, there were marked behavioral changes as a function of maternal ethanol treatment observed in the openfield and Y-maze tests. The finding of increased ambulation and rearing on the second day of open-field testing especially in G r o u p 4g offspring relative to controls in the present study lends some support to the view that the results of Bond & Di Giusto (1976, 1977) and Branchey & Friedhoff (1976, 1977) are indeed attributable to the ethanol provided dams in the liquid diet during gestation. In the Y maze, the G r o u p 6g animals made more avoidance responses than the other groups. This finding must be viewed in relation to the other measures of behavior in the task. Not only did G r o u p 6g make more avoidance responses, but it was also clearly the most active during the intertrial interval in the safe arm, at least during the initial days of testing. Further, in terms of learning the correct discrimination that was required, these animals did not differ from those in the other treatment groups. Given this pattern of results it seems reasonable to suggest that the differences in avoidance responding were related to persistent treatment-produced differences in activity in this task rather than to altered learning ability. The application of this analysis to the passive-avoidance situation would lead to the prediction that offspring of dams given ethanol during gestation would perform more poorly than offspring of control dams. The results of Riley et al. (1979) support this prediction. While there is much support for this analysis of behavior in shock-avoidance situations (e.g., Caul & Barrett, 1973; Anisman & Waller, 1971), concern with this issue should not obscure the major finding of the present study. The results clearly show that maternal ethanol treatments during days 10-14 of gestation can produce long-term behavioral changes even though not affecting growth and development of the animal. STUDY 2
Study 2 had 4 purposes. The first was to replicate the 3 ethanol treatments of the first study with the addition of an 8g/kg per day group. The second was to assess maternal blood ethanol levels. The third was to replicate Study 1 with animals derived from litters of equal size. Although the treatments of Study 1 did not affect litter size, experimental control of this variable is desirable (e.g., G r o t a & Ader, 1969). The fourth purpose was to extend the dose range and assess open-field and Y-maze performance in animals not given the extensive handling and preweaning experience necessitated by the developmental testing procedures used in the first experiment.
MATERIALS AND METHODS Nulliparous females of the same strain of rat as used in Study 1 were mated at 120 days of age. Procedures were the same as previously described with 4 exceptions. (1) Four experimental treatments were used: 8g (N = 5), 6g (N = 6), 4g (N -= 6), and 2g (N = 5). (2) Blood ethanol levels were determined for all animals from samples taken 1 hr after each intubation on days 10, 12, and 14. Animals were lightly restrained while a 10/d sample of blood was extracted from a small cut made in the tail. Ethyl Alcohol StatPacks (Calbiochem) were used for determination of blood ethanol levels. (3) Food consumption and maternal body weights were monitored as before on gestation days 10-15 but not on days 16-19 as in the previous study. (4) Litters were assessed at delivery as in Study 1, culled to 4 males and 4 females when possible, and then left undisturbed until weaning at day "28.
Open-field and avoidance performance of rats
319
RESULTS Table 2 presents food c o n s u m p t i o n and b o d y weights for the d a m s during the treatm e n t period as well as b l o o d ethanol levels. As is a p p a r e n t from the means, over the treatment period, the dose level reliably affected the a m o u n t of food eaten, F(12, 72) = 13.16, P < 0.001, and maternal b o d y weight, F(12, 72) = 50.06, P < 0.001. Analyses c o m p a r i n g the 8g and 6g g r o u p s showed that these g r o u p s were equivalent in a m o u n t of food eaten, F(1, 36) < 1, and b o d y weight, F(I, 36) < 1, during the treatment period. B l o o d ethanol levels differed as a function of treatment, F(3, 1 8 ) = 24.30, P < 0.001, ranging f r o m the mean of 238.9 mg~o in the 8g g r o u p to the mean 81.4 mg~o in G r o u p 2g. Litter assessment data as well as offspring weights a n d sample size for behavioral testing are also seen on Table 2. As in the first experiment, the treatment did n o t differentially affect the length of gestation, the n u m b e r of pups delivered, or the male/ female ratio of offspring. The addition of the 8g g r o u p in this study p r o d u c e d a reliable treatment effect on offspring b o d y weight, F(3, 242) = 92.71, P < 0.001. The m e a n pup weight for G r o u p s 8g, 6g, 4g, and 2g was 5.66g, 6.59g, 7.14g, and 6.93 g, respectively. Even t h o u g h g r o u p s 8g and 6g did not differ in maternal food c o n s u m p t i o n or b o d y weight the offspring of 8g d a m s weighed less than the offspring of 6g dams, F(1, 122) = 75.49, P < 0.001. While males were heavier than females at birth, F(1, 242) = 17.82, P < 0.001, this sex difference did not interact with treatment effects. B o d y weight at weaning on day 28 and prior to testing on day 63 was analyzed with Age as a within-subject variable to assess possible differences in g r o w t h rate. The sex difference in weight was reliable, F(1, 152) = 118.81, P < 0.001, and increased in m a g n i t u d e with age, F(1, 150) = 100.74, P < 0.001. Neither the main effect of Treatment, F(3, 152) = 2.38, P = 0.07 nor the interaction o f T r e a t m e n t x Age, F(3, 150) = 1.67, P = 0.174, was reliable. Analysis of a m b u l a t i o n scores during the 2 days of open-field testing s h o w e d that there was no evidence for an effect of maternal treatment, F(3, 101) = 1.81, P = 0.149. T r e a t m e n t effects were seen, however, with the other measures of specific behaviors. L a t e n c y to enter a new square when first placed in the o p e n field was reliably affected by Treatment, F(3, 101) = 5.00, P < 0.01, with G r o u p 8g showing the shortest latency. T h e g r o u p means were 0.95 sec for G r o u p 8g, 1.36 sec for G r o u p 6g, 1.66 sec for G r o u p 4g, and 1.29 sec for G r o u p 2g. T r e a t m e n t effects were also found for rearing, F(3, 101) = 15.07, P < 0.001, and grooming, F(3, 91) = 2.77, P < 0.05. F r e q u e n c y of each Table 2. Maternal effects, litter assessment, body weight, and sample size for behavioral testing as a function of ethanol treatment Group Mean food eaten (g)/Mean body weight (g) Gestation day 11 12 13 14 15 Mean blood ethanol level (mg~o) Number of litters delivered Median length of gestation (days) Total viable pups Total dead pups at parturition Total viable male pups Total viable female pups Mean birth weights of males (g) Mean birth weights of females (g) Day 28 mean weight of males (g) Day 28 mean weight of females (g) Day 63 mean weight of males (g) Day 63 mean weight of females (g) Males used for behavioral testing (N) Females used for behavioral testing (N)
8g 24.3/330 1.8/296 0.4/282 0.0/269 0.1/258 238.9 5 22.00 59 0 36 23 5.77 5.55 87.13 78.07 282.44 195.14 9 11
6g 23.3/320 2.8/293 0.6/276 0.5/267 0.8/262 188.7 6 22.10 67 1 34 33 6.60 6.58 82.25 77.36 286.41 207.12 15 15
4g 24.1/314 9.9/302 14.4/308 17.3/313 15.9/315 109.6 6 22.00 60 2 34 26 7.36 6.92 86.28 80.1l 307.69 214.53 14 17
2g 23.2/314 14.6/312 16.5/314 17.5/318 21.2/326 81.4 5 21.88 64 0 31 33 7.16 6.70 89.42 82.90 286.90 230.80 14 14
320
WILLIAM F. C A L L t'l al.
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of these responses was greatest in offspring of G r o u p 8g dams. The means for rearing were: 8g = 27.30, 6g = 17.15, 4g = 12.47, 2g = 16.89. The means for grooming were: 8g = 0.86, 6g = 0.68, 4g = 0.56, 2g = 0.68. Consistent with Study 1, males crossed fewer squares, F(1, 1 0 1 ) = 5.26, P < 0.05, reared less often, F(1, 101) = 5.37, P < 0.05, defecated more, F(1, 101) = 6.38, P < 0.05, and urinated more, F(1, 101) = 7.96, P < 0.01, than females. These sex differences did not in any case interact with maternal treatment. The mean number of avoidance, responses as a function of maternal ethanol dose over the 5 days of Y-maze testing is seen on Fig. 5. Analysis of these data shows that there were reliable Treatment, F(3, 101) = 3.39, P < 0.05, Days, F(4, 404) = 121.10, P < 0.001, and Treatment x Days interaction, F(12, 404) = 2.00, P < 0.05, effects. The outstanding performance of 8g offspring provided the major contribution to this result. Although the groups were ordered as a function of maternal dose with the mean avoidances per day being 8.27 for G r o u p 8g, 5.89 for G r o u p 6g, 5.39 for G r o u p 4g, and 4.77 for G r o u p 2g, the differences over days a m o n g G r o u p s 6g, 4g, and 2g were not reliable, F(8, 332) <1. Again, with regard to correct discriminations, the 8g offspring stood out with a mean per day of 17.26 relative to means of 16.63 for group 6g, 16.33 for G r o u p 4g, and 16.17 for G r o u p 2g. The effects of both Treatment, F(3, 101)= 2.85, P < 0.05, and Days, F(4, 404) = 272.03, P < 0.001, were reliable. Given this difference in discrimination performance, a Treatment difference in incorrect escapes is expected, F(3, 101) = 2.95, P < 0.05, resulting from G r o u p 8g offspring making the fewest number of incorrect escapes. The analysis of incorrect avoidance responding yielded significant Days, F(4, 404) = 9.93, P < 0.001, and Treatment x Days, F(12, 404) = 2.06, P < 0.05, factors. The means, however, were not clearly ordered by maternal dose: 8g = 0.24, 6g = 0.17, 4g = 0.27, 2g = 0.17. While intertrial activity decreased over the 5 days of Y-maze testing, F(4, 391) = 32.08, P < 0.001, this decrease was not a function of treatment group. F(12, 391) < 1. DISCUSSION
In most measures taken in the open field and Y maze, the behavior of the 8g offspring stood out. During the 2 days of open tield testing they were faster to move initially, reared more and groomed more. In the Y maze, the 8g group made the most avoidance
Open-field and avoidance performance of rats
321
responses a n d also m a d e m o r e correct d i s c r i m i n a t i o n s t h a n the pups from the other t r e a t m e n t groups. T h e findings of this second study s u p p o r t a n d extend the results of the first experiment. T h e a p p a r e n t differences in the a b s o l u t e levels of the measures t a k e n in the o p e n field a n d Y maze between the 2 studies suggest a n influence of the extensive p r e w e a n i n g h a n d l i n g a n d experience given a n i m a l s of the first study d u r i n g d e v e l o p m e n t a l testing. Nevertheless, the results of b o t h studies clearly show that b e h a v i o r at days 63-69 was modified by p r e n a t a l e t h a n o l t r e a t m e n t . G i v e n that pair fed a n d a d l i b i t u m c o n t r o l s were n o t i n c l u d e d in S t u d y 2, it is i m p o r t a n t to n o t e that the 8g a n d 6g s o l u t i o n s i n t u b a t e d were equal in calories a n d that these 2 t r e a t m e n t s had e q u i v a l e n t effects o n m a t e r n a l food c o n s u m p t i o n a n d b o d y weight, Thus, the fact that the 8g a n d 6g d a m s were n u t r i t i o n a l l y the same, suggests that the dose-response curve seen in Fig. 5 s h o w i n g a v o i d a n c e b e h a v i o r is indicative of the effects of e t h a n o l p e r se. This evidence of l o n g - t e r m b e h a v i o r a l change following p r e n a t a l exposure to e t h a n o l indicates the i m p o r t a n c e of assessing f u n c t i o n a l t e r a t o g e n i c effects. G i v e n these findings, it is n o w i m p o r t a n t to elucidate the possible a n a t o m i c a l a n d / o r b i o c h e m i c a l m e c h a n i s m s u n d e r l y i n g such b e h a v i o r a l effects which occur w i t h o u t o b v i o u s physical teratogenic effects.
REFERENCES Abel, E. L. Alcohol ingestion in lactating rats: Effects on mothers and offspring. 1. Archives Internationales de Pharmacodynamie et de Therapie, 1974, 210, 121-127. Abel, E. L. Emotionality in offspring of rats fed alcohol while nursing. Journal of Studies on Alcohol, 1975, 36, 654-658. Abel, E. L. Effects of ethanol on pregnant rats and their offspring. Psychopharmacology, 1978, 57, 5 I I. Anisman, H. & Waller, T. G. Effects of inescapable shock upon subsequent one-way avoidance learning in two strains of rats. Psychonomic Science, 1971, 24, 101 102. Barlow, S. M. & Sullivan, F. M. Behavioral teratology. In C. L. Berry & D. E. Poswillo (Eds), Teratology: Trends and Applications. New York: Springer-Verlag, 1975. Barrett, R. J., Leith, N. J. & Ray, O. S. A behavioral and pharmacological analysis of variables mediating active-avoidance behavior in rats. Journal of Comparative and Physiological Psychology, 1973, 82, 489-500. Barrett, R. J., Leith, N. J. & Ray, O. S. An analysis of the facilitation of avoidance acquisition produced by d-Amphetamine and Scopolamine. Behavioral Biology, 1974, I I, 189-203. Bolles, R. C. & Woods, P. J. The ontogeny of behaviour in the albino rat. Animal Behaviour, 1964, 12, 427M.41. Bond, N. W. & Di Giusto, E. L. Effects of prenatal alcohol consumption on open-field behaviour and alcohol preference in rats. Psychopharmqcologia (Berl.), 1976, 46, 163-165. Bond, N. W. & Di Giusto, E. L. Prenatal alcohol consumption and open-field behaviour in rats: Effects of age at time of testing. Psychopharmacology, 1977, 52, 311-312. Branchey, L. & Friedhoff, A. J. Biochemical and behavioral changes in rats exposed to ethanol in utero. Annals of the New York Academy of Sciences, 1976, 273, 328-330. Caul, W. F. & Barrett, R. J. Shuttle-box versus Y-maze avoidance: Value of multiple response measures in interpreting active-avoidance performance of rats. Journal of Comparative and Physiological Psychology, 1973, 84, 572-578. Chernoff, G. F. The fetal alcohol syndrome in mice: An animal model. Teratology, 1977, 15, 223-230. Clarren, S. K. & Smith, D. W. The fetal alcohol syndrome. New England Journal of Medicine, 1978, 298, 1063-1067. Coyle, I., Wayner, M. J. & Singer, G. Behavioral teratogenesis: A critical evaluation. Pharmacology, Biochemistry and Behavior, 1976, 4, 191-200. Grota, L. J. & Ader, R. Continuous recording of maternal behaviour in Rattus norvegicus. Animal Behaviour, 1969, 17, 722-729. Jones, K. L. & Smith, D. W. Recognition of the fetal alcohol syndrome in early infancy. Lancet, 1973, II, 999-1001. Kronick, J. B. Teratogenic effects of ethyl alcohol administered to pregnant mice. American Journal of Obstetrics and Gynecology, 1976, 124, 6764580. Lemoine, P., Harousseau, H., Borteyru, J.-P. & Menuet, J.-C. Les enfants de parents alcooliques: anomalies observ6es ~. propos de 127 cas. Quest Medical, 1968, 25, 47~482. Morra, M. Ethanol and maternal stress on rat offspring behaviors. Journal of Genetic Psychology, 1969, i14, 77-83. Osborne, G. L. & Caul, W. F. Y-maze avoidance performance and activity change as a function of CS quality in rats. Behavioral Biology, 1975, 15, 183-192. Randall, C. L. Teratogenic effect of in utero ethanol exposure. In K. Blum (Ed.), Alcohol and Opiates: Neuroehemical and Behavioral Mechanisms. New York: Academic Press, 1977. Randall, C. L., Taylor, W. J. & Walker, D. W. Ethanol-induced malformations in mice. Alcoholism Clinieal and Experimental Researeh, 1977, I, 219 223.
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Riley, E. P., Lochry, E. & Shapiro, N. Lack of response inhibition in rats prenatally exposed to alcohol. Psychopharmaeology, 1979, In press. Rosenblatt, J. S. & Lehrman, D. S. Material behavior of the laboratory rat. In H. S. Reingold (Ed.), Maternal Behavior of Mammals. New York: Wiley, 1963. Rosett, H. L. Maternal alcoholism and intellectual development of offspring. Lancet, 1974, 2, 218. Streissguth, A. P. Psychologic handicaps in children with the fetal alcohol syndrome. Annals of the New York Academy of Science, 1976, 273, 14(~145. Streissguth, A. P., Herman, C. S. & Smith, D. W. Intelligence, behavior and dysmorphogenesis in the fetal alcohol syndrome: a report on 20 patients. Journal of Pediatrics, 1978, 92, 363 367. Tze, W. J. & Lee, M. Adverse effects of maternal alcohol consumption on pregnancy and foetal growth in rats. Nature, 1975, 257, 479-480. Ulleland, C. W. The offspring of alcoholic mothers. Annals ~?["the New York Academy o.1 Sciences, 1972, 197, 167-169. Vincent, N. M. The effects of prenatal alcoholism upon motivation, emotionality and learning. American Psychologist, 1958, 13, 401. Vorhees, C. V. Some behavioral effects of maternal hypervitaminosis A in rats. Teratology, 1974, 10, 269-273. Vorhees, C. V., Barrett, R. J. & Schenker, S. Increased muricide and decreased avoidance and discrimination learning in thiamine deficient rats. Life Sciences, 1975, 16, 1187-1200. Werboff, J. & Gottlieb, J. S. Drugs in pregnancy: Behavioral teratology, Obstetrics and Gynecology Survey, 1963, 18, 420-423. Wilson, J. G. Environment and Birth Defects. New York: Academic Press, 1973. Wilson, J. G. Critique of current methods for teratogenicity testing in animals and suggestions for their improvement. In T. H. Shepard, J. R. Miller & M. Marois (Eds), Methods for Detection of Environmental Agents That Produce Congenital Dejects. New York: Elsevier, 1975. Yanai, J. & Ginsburg, B. E. Audiogenic seizures in mice whose parents drank alcohol. Journal of Studies on Alcohol, 1976, 37, 1564--1571.