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Effect of moderate prenatal ethanol exposure on postnatal brain and behavioral development in BALBc mice
EXPERIMENTAL
NEUROLOGY
89,237-249
(1985)
Effect of Moderate Prenatal Ethanol Exposure on Postnatal Brain and Behavioral Development in BALB/c Mice PATRICIA WAINWRIGHT AND GISELA FRITZ’ Deparlment of Health Studies, University of Waterloo, Waterloo, Ontario NZL 3G1, Canada Received January 3, 1985 Prior research had indicated that moderate maternal ethanol consumption during gestation affected the growth of the corpus callosum and anterior commissure in BALB/c mice when measured at day 19 postconception. Our purpose was to assess whether or not this was an enduring effect. Pregnant BALB/cCRBL mice were fed ethanol 10% v/v in the drinking water from days 5 to 26 postconception. Control animals received an isocaloric sucrose solution and were pair-fed to the experimental animals. An additional control group fed laboratory chow ad libitum was included. Using a split-litter design, brain development was assessed on days 26 and 50 postconception and behavioral development of the pups was measured on day 32. The ethanol-treated offspring had lower brain weights at both ages as well as a smaller cross-sectional area of the anterior commissure on day 50, which was significantly related to the smaller brain weight. There was no apparent effect of ethanol on the area of the corpus callosum at either age. Similarly, behavioral development was not affected by the treatment, although eye-opening was delayed in ethanol-treated animals. Measures of maternal behavior indicated that the animals consuming alcohol were more active than those in the control groups. An unexpected finding was that the control group fed sucrose appeared to be adversely affected. The body weight of these pups was lower, as was the area of the corpus callosum at day 50.
0 1985 Academic
Press, Inc.
INTRODUCTION
Prenatal ethanol exposure in humans has been associated with a characteristic pattern of fetal defects collectively termed the fetal alcohol syndrome Abbreviations: CC-corpus callosum, CA-anterior commissure. ’ This study was completed in partial fulfillment of the requirements for the M.Sc. degree in Health Studies by G.F. The authors thank Mr. W. Zagaja for animal maintenance, Ms. M. Gagnon and Mrs. K. Koenderink for technical assistance, and Ms. S. Hurlburt for secretarial support. In addition Dr. D. DiBattista, Dr. 0. Martinez, and Dr. D. Mills provided helpful advice. The research was supported by Natural Sciences and Engineering Research Council of Canada grant A76 I7 to Patricia Wainwright. 237 0014-4886185 $3.00 Copyright 8 1985 by Academic Press. Inc. All ri&ls of reproduction in any form reserved
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(FAS) (1). Both human and animal studies have indicated that ethanol adversely affects the development of the central nervous system; doses that do not produce overt teratology may nevertheless cause developmental delay and alter the organization of neuronal networks (1, 22). Because of reports in the literature suggesting that prenatal ethanol exposure might be associated with agenesis of the corpus callosum (CC) (4, 5), a recent study was conducted to determine whether or not moderate consumption of ethanol during gestation would increase the incidence of CC defects in BALB/c mice ( 19). This strain is prone to deficiency of the CC, where in some animals the forebrain commissural tract is either missing at midline or is greatly reduced in size (12). The results of the study did not support an increased incidence of callosal agenesis, but showed that the growth of both the CC and anterior commissure (CA) was affected by the ethanol treatment; the effect appeared to be independent of the smaller brain weight in those animals. This finding contrasted with the observation that there was no apparent effect of the same regimen of ethanol exposure on brain development in C57BL/6 mice. As these effects were observed in IPdayold BALB fetuses, the question still remains of whether they reflected a developmental delay that would be amenable to “catch-up” growth or whether ethanol has a permanent effect on the growth of these fiber tracts. Our purpose was to resolve this issue by allowing the animals to develop beyond the period of ethanol administration to determine if the treatment produced enduring effects. The ethanol was administered to the animals in their drinking water, which was their sole source of fluid. The cross-sectional area of the CC and CA were measured in a midsagittal section on days 26 and 50 postconception. In addition, maternal behavior was assessed on day 26 and behavioral development of the pups was measured on day 32 postconception, using a standardized behavioral developmental scale (13, 14). METHOD Subjects. Male and virgin female BALB/c mice were purchased from Charles River Breeding Laboratories, St. Constant, Quebec, at 35 days of age. They were housed in groups of four to five in standard opaque plastic mouse cases with Beta-Chip bedding and several sheets of toilet tissue for nesting. They were allowed free access to laboratory chow (Master Rodent Cubes, Maple Leaf Mills, Toronto) and tap water. They were maintained at 22 + 1 “C and the 12-h dark:lZh light cycle was reversed, with dark from 0800 to 2000 h. Breeding commenced when the animals were 60 days old; 71 pregnant females were randomly assigned to the three treatment groups. In the alcohol-treated group, 7/23 gave birth to live pups; in the pair-fed control group, 7/3 1; and in the untreated control group, 6/ 17.
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Experimental Design. Inasmuch as ethanol consumption has been shown to depress food intake in addition to supplying “empty” calories, it was important to control for a possible nutritional confounding of the results. To this end, two control groups were used; one, the untreated control, had access to food and water ad libitum and in the other, the pair-fed control group, each animal was matched by weight to an animal receiving alcohol and was fed the same amount of food and volume of fluid ingested by it on the same day of pregnancy. In the latter group the calories derived from alcohol were replaced by an isocaloric sucrose solution. Comparison between the two control groups allowed assessment of the nutritional effect of the ethanol treatment. The three groups therefore consisted of animals with (i) unrestricted access to 10% ethanol (v/v) in the drinking water and to laboratory chow, (ii) pair-feeding of an isocaloric sucrose solution and laboratory chow on the basis of a match in body weight to the animals in (i), and, (iii) unrestricted access to drinking water and laboratory chow. Materials. The alcohol was purchased from the Liquor Control Board of Ontario and was supplied as a 40% (v/v) solution, known commercially as Alcool. This was diluted with tap water to 10% (v/v), equivalent to 0.56 kcal/ml. The concentration of the isocaloric sucrose solution was 14% (v/v) in tap water. The blood alcohol concentrations were determined by the alcohol dehydrogenase method provided as a diagnostic kit by Sigma Chemicals (Sigma Technical Bulletin No. 332-4V). Procedure. Animals were mated at the onset of the dark cycle and after 6 h the male was removed and the female examined for the presence of a vaginal plug. The day a plug was detected was designated day 0 of gestation and all days, both pre- and postnatal, were counted from that point. The mice were randomly assigned to ethanol treatment or control groups, although within those assigned as controls the pair-fed animals were selected on the basis of their match in body weight to alcohol-treated animals. Because of difficulties in maintaining pregnancies among the sucrose-treated dams, three of them were pair-fed with a partner receiving alcohol, even though their body weights were 20% higher. In this case food intake was adjusted so that intake per gram body weight remained equivalent. The body weight of all maternal animals was recorded on days 0, 5, 12, 19, 22, 32, and 50. On day 50 the dams were killed and the brains removed, fixed in 10% Formalin, and blotted and weighed within 1 week. All pups were counted at birth and gestation length and litter size were recorded. On day 22, pups were weighed and maternal behavior tested with four pups in the home cage. (The remaining pups were kept warm under an infrared heat lamp.) Because experiential factors during early development have been found to influence development (6), this test was done to ascertain whether or not the dam’s behavior might have been affected by
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the alcohol treatment and thereby influence the development of the pups. The behavioral test consisted of a lo-min observation period during which each dam’s activity was recorded every 5 s, including the time when the first and last pups were retrieved; an electronic beeper was used as a timer. The activities were recorded in the following 20 mutually exclusive behavioral categories. Climb-quadrupedal motion along the horizontal wire grid of the cage lid. Run-walk-quadrupedal movement on the floor of the cage, the run distinguished by its relatively higher speed. Air sniff-animal stationary and sniffing with the nose in the air, while all four legs are on the ground, or with one or both forepaws raised or leaned on. Sniff object or pup-animal stationary and sniffing at the bedding or nesting material (object) or at the pup. Nuzzle pup-animal stationary while sniffing vigorously at pup, often pushing it with the nose and pawing it with the forepaws. Carry pup/nesting material-walking or running while grasping the pup or nesting material between teeth with head slightly raised. Pull/shred paper-sitting in one place with paper in teeth and jerking it around sideways, sometimes accompanied by chewing the edges of the paper. In nest-animal is in the nest, but obscured from view by nesting material. Arrange-sitting in the nest and pushing the nesting material in all directions with forepaws. Retrieve pups-transport of pups back to the nest after the experimenter has placed them in the corner opposite the nest. Rick dig-scraping the bedding material under the abdomen with alternating movements of forepaws, and then moving it farther backward kicking both hind legs; sometimes digging with only the forepaws is seen. Push dig-pushing the bedding material in front with alternating movements of forepaws accompanied by running. Push dig usually occurs after a sequence of kick dig, with the result that the material removed while digging a hollow is piled to one side of the cage. Groom-attending to any part of its own body, by wiping with the forepaws (usually the head), licking, scratching with the hind paws, or nibbling. Eat-chewing and swallowing pieces from a food pellet, which may be held between the forepaws. Drink-swallowing water obtained by licking at the spout of the drinking bottle.
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Although all behavioral testing was carried out by the same experimenter who administered the treatment, precautions, such as obscuring the identification tags, were taken to preserve objectivity. Because the CC in this strain attains the adult range of size about 1 week after birth (15), by measuring the structure on day 26 the possibility of a ceiling effect was minimized. Therefore, on day 26 all pups were weighed and sexed, and half the litter from each sex was chosen randomly, anesthetized with chloroform, and perfused intracardially with physiologic saline followed by 10% buffered Formalin. The brains were exposed by removal of the skull cap and suspended 24 h in 10% Formalin before being removed. They were trimmed by cutting vertically through the brain stem at the base of the cerebellum, blotted, and weighed. The brains were then embedded in gelatin, sectioned in the sag&al plane at 33 pm, and stained with hemotoxylin and eosin. The cross-sectional areas of the CC and CA were determined from tracings of midsagittal sections as described elsewhere (17). All viewing was done without prior knowledge of the experimental status of the tissue. Starting on the morning of day 26, the remaining animals were gradually weaned off alcohol until day 3 1, when they were receiving only water. On day 32 the pups were weighed and their behavioral development tested as follows: Righting reflex-does S return rapidly to its feet when placed on its back? Cliff aversion-does S withdraw from the edge of a flat surface when its snout and forepaws are placed over the precipice? Forelimb and hind limb grasp reflex-does S grasp strongly the barrel of an 18-gauge needle when it is touched to the palm of each forepaw (hind paw)? Vibrissa placing reflex-does S place its forepaw onto a cotton swab stroked across its vibrissae? Level screen test-can S hold onto a piece of 288-mesh aluminum screen when S is dragged across it horizontally by the tail? Vertical screen test-can S hold onto the screen when this is placed vertically? Screen climbing test-can S climb the vertical screen using both foreand hind limbs? Pole grasp-can S grasp the shaft (2.5 mm) of a cotton swab firmly with both fore- and hind paws? Forelimb and hind limb stick grasp-can S grip firmly a 9.5-mm-wide wooden stick with forepaws (hind paws)? Eyes open-are both eyes fully open? Visual placing reflex-does S extend its forelimbs when its is lowered rapidly toward a flat surface?
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Auditory startle response-does S show a whole-body startle response when a loud snap of fingers occurs less than 15 cm away? Each pup was assigned a score between 0.0 and 1.0 on each measure. It was possible to standardize the composite score of the animal in terms of behavioral age by substituting it into the regression equation y = 24.40 + 11.14x - 1.092 where y = age and x = score. This equation was derived by testing separate litters of a standard B6D2FI/B6D2FI hybrid cross on the behavioral test battery on each of gestation days 27 through 36 and reflects the relationship in this population between chronologic age and the litter mean behavioral scores (14). Using this scale, inbred animals were shown to be retarded in behavioral development, as were animals that were malnourished during gestation (16, 18). Litters were monitored daily and the day recorded when all animals in a litter had open eyes. On day 50 the dams and pups were weighed and their body lengths measured. They were then anesthetized with an overdose of sodium pentobarbital and perfused with physiologic saline and 10% buffered Formalin. The brains were removed and weighed as described above. Frozen sections of the brain were stained with metachromatic thionin and the CC and CA measured as described. Blood alcohol concentrations were measured on a separate but comparable group of pregnant animals between days 14 and 18 of gestation between 1400 and 1700 h. Blood was removed from the heart into a heparinized vial and stored for 24 to 48 h prior to analysis. Analysis. The data were analyzed using the GLM (general linear model) provided by SAS (Statistical Analysis System) for analysis of variance. Because of the hierarchical nature of the design, i.e., litters nested within treatment groups, the between-litter variance was used as the error term. Groups means were compared by preplanned t tests; the significance level was set at (r = 0.05. RESULTS Pup Variables. The data on the variables related to the pups are presented
in Table 1. Body weight. On each of days 22, 26, 32, and 50, the pups of the untreated control group had the highest body weight, and although the ethanol-treated pups weighed less than these, the differences were not statistically significant. Surprisingly, the pups of the pair-fed control group weighed less than either of the other two groups and were significantly lighter than the pups of the untreated control group. The same results held true of body length on day 50.
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TABLE 1 Effect of Ethanol Consumed from Day 5 to Day 26 Postconception on Pup Variables in BALB/c Mice0 Ad libitum-fed
Days postconception MY
wwt
Alcohol-fed (7)
Pair-fed sucrose (7)
lab chow
(6)
(9)
22 26 32 50
2.01 3.87 7.80 15.64
k 0.03 kO.15 + 0.20 + 0.31
1.89 3.36 6.99 14.89
+ + f k
0.04* 0.15* 0.19* 0.40”
2.15 4.10 8.20 16.31
+ 0.11*
8.26
+ + f +
0.04’ 0.13c 0.20’ 0.32’
Body length (cm) 50
7.96
It: 0.07
7.79
Brain weight (g) 26 50
0.2548 2 0.005” 0.4901 f 0.004*
0.2766 + 0.007 0.5012 + 0.006
0.2801 f 0X107~ 0.5104 + 0.003’
Corpus callosum length (mm) 26 50
2.321 k 0.176 3.368 zk 0.055<
2.048 -t 0.175 3.032 + 0.177”
2.266 + 0.164 3.412 + 0.093’
Corpus callosum ares (mm 2, 26 50
0.7786 + 0.059 1.0259 * 0.017
0.6665 k 0.056 0.9194 + 0.052*
0.7998 + 0.056 1.0914 + 0.028’
Anterior commissure (mm 2, 26 50
0.1109 & 0.003 0.1368 f 0.006*
0.1110 + 0.003 0.1551 * 0.006
0.1199 + 0.005 0.1576 + 0.005’
Behavioral scored 32
0.62
+ 0.02
0.63
+ 0.02
0.62
f 0.02
Eye opening (days)
35.00
f 0.446
34.29
f 0.29
33.50
+ 0.22E
+ 0.07c
’ Data are presented as f & SE. Parentheses show number of litters. ** Means with different superscripts in the same row are significantly different, P < 0.05. d Behavioral age Q = 30.9 days. CalcuIated by substituting behavioral score (x) into equation y = 24.40 + 11.14-x - 1.09x2.
Bruin weight. On both days 26 and 50, in pups of the ethanol-treated group the brains weighed significantly less than those of the untreated control group (one-tailed test) whereas the pair-fed control group did not differ significantly from either group. These results do not covary with those on body weight, suggesting that brain growth was affected independently of differences in body weight between the groups.
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Corpus callosum. On day 26 there were no significant differences among the groups in the area of the CC, but on day 50 this variable was significantly smaller in the pair-fed control group than in the untreated control group. The alcohol-treated group was not significantly different from either of the control groups. A similar pattern of results was seen for CC length, except on day 50 when the pair-fed control differed from both other groups. Only two pups in the study were found to be acallosal at day 50 and these originated from one pair-fed control litter; they were not included in further analysis. The effects on the corpus callosum do not reflect the same betweengroup differences seen in brain weight, where the brains of the ethanol groups weighed the least, indicating that the effect in the pair-fed control groups was independent of an effect on overall brain growth. This is supported by the fact that on both days 26 and 50 there was no significant linear correlation between brain weight and CC area in the untreated control group (day 26, r2 = 0.0034; day 50, r2 = 0.0032; P > 0.05). Anterior commissure. Again there were no significant differences among the groups on day 26, but on day 50 the mean area of the CA in pups of the alcohol-treated group was significantly smaller than that of the untreated control group. This effect appeared to be related to the reduced brain weight in these animals inasmuch as the ratio of CA area to brain weight was not significantly different among the groups. Behavioral score. There was no indication of behavioral retardation due either to inadequate nutrition or the consumption of ethanol. All groups had a behavioral age of 30.9 days, which is within the range of scores expected of inbred animals (16). However, pups of dams treated with ethanol opened their eyes an average of 1.5 days later than those of the untreated control group. Maternal Variables. The data on the variables related to the dams are presented in Table 2. Food and fluid consumption. Caloric intake did not differ between the groups of animals that consumed ethanol and the untreated control group. During the 3rd week, intake of the pair-fed control group was lower than the ad libitum-fed group, reflecting the fact that on some occasions the mice did not eat their entire ration of food. However, fluid intake during the last 2 weeks of treatment was significantly lower in the experimental group, which consumed an average of 20% of their calories as ethanol in the 1st week of treatment, decreasing to 18% during the next 2 weeks. This is equivalent to 16 g/kg ethanol per day, and should be considered in relation to a metabolic rate of alcohol elimination of 550 mg/kg/h in the mouse, compared with 300 mg/kg/h in rat and 100 mg/kg/h in man (21). This may partially explain the low blood alcohol concentrations we observed (4.58 f 0.64 mg/lOO ml, N = 6).
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TABLE 2 Effect of Ethanol Consumption from Day 5 to Day 26 Postconception on Maternal Variables in BALB/c Mice’ Ad libitum-fed
Days postconception Body weight (g) 5 12 19 22 26 32 50
Alcohol-fed (7) 20.71 23.54 34.17 24.27 24.64 25.89 24.37
+ f f f f + f
0.43’ 0.16 1.14 0.31 0.49’ 0.44’ 0.39’
Pair-fed sucrose (7) 22.74 23.70 32.53 23.10 23.13 25.17 26.51
+ k f f + + +
0.53’ 0.52 1.80 0.62* 0.55* 1.03’ 0.58’
lab chow
(6) 21.33 24.62 35.28 25.52 27.50 28.33 25.83
+ + k + + + +
0.51 0.69 1.51 0.75’ 0.68’ 0.76’ 0.33’
Brain weight (g) 50
0.5023 3~ 0.012*
0.5394 + 0.010’
0.5267 + 0.004
Litter size 0 22 26
5.29 4.86 4.57
f 0.75* f 0.70* zk 0.61
7.29 6.86 5.43
f 0.36’ + 0.51C + 0.48
5.67 5.50 5.50
+ 0.61 + 0.62 f 0.62
Gestation length (days)
20.29
+ 0.18*
19.86
+ 0.14’
20.00
+ 0.00
Maternal age (days)
78.86
+ 3.92*
100.29
2 2.52’
86.67
+ 6.22
Maternal behavior Locomote Air sniff Drink
28.57 45.57 0.14
f 2.83* * 4.57” + o.14c
18.86 24.00 9.71
f 5.14 k 2.51’ f 1.666
13.33 f 4.70c 31.67 + 6.35’ 1.67 + 1.67’
91.98 118.64 147.23
+ 4.38 f 2.58 * 6.94
85.88 110.86 132.48
f 1.59 _t 2.10 + 5.49”
85.68 122.00 168.02
k 4.88 _t 7.17 + 11.19’
Fluid intake (ml) 5-11 12-18 19-26
34.23 40.31 48.74
f 1.42 + 1.736 f 3.90*
37.89 45.19 56.83
f 2.39 zk 3.94 + 6.17
39.53 52.37 71.97
+ 1.29 f 2.23’ + 4.52’
% Ethanol-derived calories 5-11 12-18 19-26
20.55 18.71 18.13
Z!T0.50 k 0.79 f 0.89
4.58
+ 0.64
Caloric intake (kcal) 5-11 12-18 19-26
Blood alcohol wncentration (mg/lOO ml)
o Data are presented as X + SE. Parentheses show number of litters. bSMeans with different superscripts in the same row are significantly different, P < 0.05.
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Maternal age. The dams in the pair-fed control group were an average 22 days older than the ethanol-treated group, a statistically significant difference in age. The reason for this difference was that many animals initially assigned to the group fed sucrose failed to maintain their pregnancies, and therefore had to be replaced later. The untreated control group did not differ in average age from either of the other two groups. Body weight. At the beginning of treatment on day 5, the dams in the pair-fed control group weighed significantly more than those in the ethanoltreated group, although neither differed from the untreated control group. This can be explained by the age differences reported above. There were no differences at days 12 and 19 during gestation, but on day 22 the animals in the pair-fed control weighed significantly less than those in the untreated control group; on days 26 and 32 both the pair-fed control and the ethanoltreated animals weighed significantly less than this control group and on day 50 the ethanol-treated group weighed significantly less than either control group. Brain weight. The brains of the dams that had consumed ethanol weighed significantly less than those of the pair-fed control group. Litter size. At birth and day 22, the ethanol-treated dams had significantly smaller litters than those of the pair-fed control group; by day 26 this difference was no longer apparent due to the fact that many sucrose-fed pups died during that period. Gestation length. The ethanol treatment led to a gestation period almost 0.5 days longer than that in the pair-fed control group. Maternal behavior. The behavioral categories of climbing, running, and walking were combined into a single “locomotion” variable, and the alcohol-treated dams had significantly higher scores than the untreated control group. Air sniffing also increased significantly in the alcohol group compared with both control groups. The animals in the pair-fed control group had significantly higher drinking scores than those in the other two groups, a reflection of the pair-feeding procedure in which the sucrose-fed animals consumed all their rations within a short time after being fed. DISCUSSION The results of this study showed that exposure to ethanol from days 5 to 26 postconception caused a decrease in average brain weight on day 26 and a decrease in both average brain weight and average CA area on day 50. The effect on the area of the CA was, however, not independent of the effect on brain weight. Ethanol caused neither an increase in the number of acallosal animals nor an effect on callosal growth. This finding is in
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contrast with the results of the earlier study (19) in which a similar regimen of ethanol exposure resulted in a smaller cross-sectional area of the CC on day 19 postconception. One possible explanation for this difference might be that the two studies used two different sublines of BALB/c mice; those in the first study were BALB/cCF whereas the present study used BALB/ cCRBL. Another difference is that in the first study the alcohol intake ranged between 20 and 22% of the dietary calories, whereas in the present study it was between 18 and 20%. It is doubtful, however, that this small difference could account for the discrepancy in outcome. An alternative explanation might be that when the CC is growing rapidly as on day 19, a small developmental delay will cause a significant difference in size, but by day 26, when growth will have slowed down, the effects will not be as dramatic. It should be noted that five brains, all from alcohol-treated animals, disintegrated before they could be examined histologically, which may lend a conservative bias to the results. It appears that the ethanol treatment also affected the adult brains; 3 weeks after cessation of treatment these dams had significantly smaller brain weights than the sucrose-treated controls. This may be further evidence for neuropathology caused by chronic intake of ethanol (8). What is interesting about our results is the unexpected effect on the growth of the offspring in the pair-fed sucrose group. Body weight was significantly smaller in this group than in the untreated control group at all ages, and although there was no effect on brain weight, the cross-sectional area of the CC was significantly reduced at day 50. One female pup in this group showed anomalous development of the pelvis and tail; this is of interest because sacral-coccygeal defects have been reported to a much higher degree in the offspring of diabetic women (11). Similarly, maternal weight on day 22 showed that this group weighed the least, suggesting that the animals may have been unable to utilize the calories provided as sucrose. It has been shown that hyperahmentation with hypertonic glucose solutions may interfere with insulin regulation and thereby engender hyperglycemia (3). A recent study (2) found that the offspring of diabetic rats were markedly smaller in birth weight than their controls; raising questions about whether or not sucrose is the appropriate control for the empty calories provided by ethanol; certainly our results suggest that a high intake of sucrose may itself be deleterious to growth. In this respect it is interesting to note recent data that also suggest deleterious effects of a sucrose diet on reproduction and survival in rodents (9). The dosage of alcohol used in this study did not affect the behavioral development of the pups, although eye-opening was delayed, which has been observed in previous studies in rats ( 10) with a trend being evident in
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mice (7). The ethanol-treated mothers showed increased activity, which replicates previous findings indicating that small doses of ethanol cause an increase in locomotor and exploratory activity (20). In summary, the results of this study indicated that moderate ethanol consumption from days 5 to 26 postconception resulted in smaller brains measured at day 50 postconception. The area of the CA was also smaller, but only in relation to the smaller brain weight. Contrary to expectation, there was no observable effect of ethanol on the growth of the CC although there did appear to be an adverse effect due to the sucrose treatment. These findings do not support the hypothesis that moderate ethanol treatment has an enduring effect on the growth of these two forebrain fiber tracts. REFERENCES I. ABEL, E. L. (Ed.). 1984. Fetal Alcohol Syndrome and Fetal Alcohol Effects Plenum, New York. 2. AERTS, L., AND F. A. VAN ASSCHE, 198 1. Endocrine pancreas in the offspring of rats with experimentally induced diabetes. J. Endocrinol. 88: 8 l-88. 3. BERDANIER, C. D. AND M. V. KAMINSKI. 1976. Carbohydrate nutrition and hyperalimentation. Pages 129-145 in C. D. BERDANIER, Ed., Advances in Modern Nutrition, Vol. 1. Wiley, New York. 4. CHERNOFF, G. 1977. The fetal alcohol syndrome in mice: an animal model. Teratology 15: 223-230.
5. CLARREN, S. K., E. C. ALVORD, JR., S. M. SUMI, A. P. STREISGUTH, AND D. W. SMITH. 1978. Brain malformations related to prenatal exposure to ethanol. J. Pediatr. 92: 6467. 6.
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CRNIC, L. B. 1983. Effects of nutrition and environment on brain biochemistry and behavior. Dev. Psychobiol. 16: 129-145. EWART, F. G., AND M. G. CUTLER, 1979. Effects of ethyl alcohol on development and social behavior in the offspring of laboratory mice. Psychopharmacology 62: 247-25 1. LIEBER, C. S. (Ed.). 1982. Medical Disorders of Alcoholism: Pathogenesis and Treatment. Saunders, Philadelphia. SHAFRIR, E., AND J. H. ADLER. 1984. Effect of long-term sucrose diet on the reproduction and survival of spiny mice (Acornys cahirinus). Nut. Res. 4, 495-501. SHAYWITZ, B. A., G. G. GRIFFIETH, AND J. B. WARSHAW. 1979. Hyperactivity and cognitive deficits in developing rat pups born to alcoholic mothers: an experimental model of the expanded fetal alcohol syndrome (EFAS) Neurobehav. Toxicol. 1: 113122. SOLER, N. G., C. H. WALSH, AND J. M. MALINS. 1976. Congenital malformations in infants of diabetic mothers. Q. J. Med. 178, 303-313. WAHLSTEN, D. 1974. Heritable aspects of anomalous myelinated fiber tracts in the forebrain of the laboratory mouse. Brain Res. 68, l-18. WAHLSTEN, D. 1974. A developmental time scale for postnatal changes in brain and behavior of B6D2F, mice. Brain Res. 72: 25 l-264. WAHLSTEN, D. 1975. Genetic variation in the development of mouse brain and behavior: evidence from the middle postnatal period. Dev. Psycho&o/. 8: 371-380. WAHLSTEN, D. 1984. Growth of the mouse corpus callosum. Dev. Brain. Res. 15: 59-67.
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16. WAINWRIGHT, P. 1980. Relative effects of maternal and pup heredity on postnatal mouse development. Dev. P&&o/. 13: 493-498. 17. WAINWRIGHT, P., AND R. STEFANESCU. 1983. Prenatal protein deprivation increases defects of the corpus callosum in BALB/c laboratory mice. Exp. Neural. 81: 694-702. 18. WAINWRIGHT, P., AND V. RUSSELL. 1983, A quantitative assessment of the effects of mahmtrition on behavioral development on laboratory mice. Behav. Neural. Biol. 39: 140-144. 19. WAINWRIGHT, P., AND M. GAGNON. 1985. Moderate prenatal ethanol exposure interacts with strain in affecting brain deveIopment in BALBfc and C57BL/6 mice. Exp. Neural., in press. 20. WALDECK, B. 1974. Ethanol and caffeine: a complex interaction with respect to locomotor activity and central catecholamines. Psychopharmacology 36: 209-220. 21. WALLGREN, H., AND H. BARRY (I&.). 1970. Actions of Alcohol, Vol. 1, Biochemical Physiological and Psychological Aspects. Elsevier, Amsterdam. 22. WEST, J. R., AND C. A. HODGES-SAVOLA. 1983. Permanent hippocampal mossy fiber hyperdevelopment following prenatal ethanol exposure. Neurobehav. Toxicol. Teratol. 5: 139-150.
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Effect of Moderate Prenatal Ethanol Exposure on Postnatal Brain and Behavioral Development in BALB/c Mice PATRICIA WAINWRIGHT AND GISELA FRITZ’ Deparlment of Health Studies, University of Waterloo, Waterloo, Ontario NZL 3G1, Canada Received January 3, 1985 Prior research had indicated that moderate maternal ethanol consumption during gestation affected the growth of the corpus callosum and anterior commissure in BALB/c mice when measured at day 19 postconception. Our purpose was to assess whether or not this was an enduring effect. Pregnant BALB/cCRBL mice were fed ethanol 10% v/v in the drinking water from days 5 to 26 postconception. Control animals received an isocaloric sucrose solution and were pair-fed to the experimental animals. An additional control group fed laboratory chow ad libitum was included. Using a split-litter design, brain development was assessed on days 26 and 50 postconception and behavioral development of the pups was measured on day 32. The ethanol-treated offspring had lower brain weights at both ages as well as a smaller cross-sectional area of the anterior commissure on day 50, which was significantly related to the smaller brain weight. There was no apparent effect of ethanol on the area of the corpus callosum at either age. Similarly, behavioral development was not affected by the treatment, although eye-opening was delayed in ethanol-treated animals. Measures of maternal behavior indicated that the animals consuming alcohol were more active than those in the control groups. An unexpected finding was that the control group fed sucrose appeared to be adversely affected. The body weight of these pups was lower, as was the area of the corpus callosum at day 50.
0 1985 Academic
Press, Inc.
INTRODUCTION
Prenatal ethanol exposure in humans has been associated with a characteristic pattern of fetal defects collectively termed the fetal alcohol syndrome Abbreviations: CC-corpus callosum, CA-anterior commissure. ’ This study was completed in partial fulfillment of the requirements for the M.Sc. degree in Health Studies by G.F. The authors thank Mr. W. Zagaja for animal maintenance, Ms. M. Gagnon and Mrs. K. Koenderink for technical assistance, and Ms. S. Hurlburt for secretarial support. In addition Dr. D. DiBattista, Dr. 0. Martinez, and Dr. D. Mills provided helpful advice. The research was supported by Natural Sciences and Engineering Research Council of Canada grant A76 I7 to Patricia Wainwright. 237 0014-4886185 $3.00 Copyright 8 1985 by Academic Press. Inc. All ri&ls of reproduction in any form reserved
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(FAS) (1). Both human and animal studies have indicated that ethanol adversely affects the development of the central nervous system; doses that do not produce overt teratology may nevertheless cause developmental delay and alter the organization of neuronal networks (1, 22). Because of reports in the literature suggesting that prenatal ethanol exposure might be associated with agenesis of the corpus callosum (CC) (4, 5), a recent study was conducted to determine whether or not moderate consumption of ethanol during gestation would increase the incidence of CC defects in BALB/c mice ( 19). This strain is prone to deficiency of the CC, where in some animals the forebrain commissural tract is either missing at midline or is greatly reduced in size (12). The results of the study did not support an increased incidence of callosal agenesis, but showed that the growth of both the CC and anterior commissure (CA) was affected by the ethanol treatment; the effect appeared to be independent of the smaller brain weight in those animals. This finding contrasted with the observation that there was no apparent effect of the same regimen of ethanol exposure on brain development in C57BL/6 mice. As these effects were observed in IPdayold BALB fetuses, the question still remains of whether they reflected a developmental delay that would be amenable to “catch-up” growth or whether ethanol has a permanent effect on the growth of these fiber tracts. Our purpose was to resolve this issue by allowing the animals to develop beyond the period of ethanol administration to determine if the treatment produced enduring effects. The ethanol was administered to the animals in their drinking water, which was their sole source of fluid. The cross-sectional area of the CC and CA were measured in a midsagittal section on days 26 and 50 postconception. In addition, maternal behavior was assessed on day 26 and behavioral development of the pups was measured on day 32 postconception, using a standardized behavioral developmental scale (13, 14). METHOD Subjects. Male and virgin female BALB/c mice were purchased from Charles River Breeding Laboratories, St. Constant, Quebec, at 35 days of age. They were housed in groups of four to five in standard opaque plastic mouse cases with Beta-Chip bedding and several sheets of toilet tissue for nesting. They were allowed free access to laboratory chow (Master Rodent Cubes, Maple Leaf Mills, Toronto) and tap water. They were maintained at 22 + 1 “C and the 12-h dark:lZh light cycle was reversed, with dark from 0800 to 2000 h. Breeding commenced when the animals were 60 days old; 71 pregnant females were randomly assigned to the three treatment groups. In the alcohol-treated group, 7/23 gave birth to live pups; in the pair-fed control group, 7/3 1; and in the untreated control group, 6/ 17.
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Experimental Design. Inasmuch as ethanol consumption has been shown to depress food intake in addition to supplying “empty” calories, it was important to control for a possible nutritional confounding of the results. To this end, two control groups were used; one, the untreated control, had access to food and water ad libitum and in the other, the pair-fed control group, each animal was matched by weight to an animal receiving alcohol and was fed the same amount of food and volume of fluid ingested by it on the same day of pregnancy. In the latter group the calories derived from alcohol were replaced by an isocaloric sucrose solution. Comparison between the two control groups allowed assessment of the nutritional effect of the ethanol treatment. The three groups therefore consisted of animals with (i) unrestricted access to 10% ethanol (v/v) in the drinking water and to laboratory chow, (ii) pair-feeding of an isocaloric sucrose solution and laboratory chow on the basis of a match in body weight to the animals in (i), and, (iii) unrestricted access to drinking water and laboratory chow. Materials. The alcohol was purchased from the Liquor Control Board of Ontario and was supplied as a 40% (v/v) solution, known commercially as Alcool. This was diluted with tap water to 10% (v/v), equivalent to 0.56 kcal/ml. The concentration of the isocaloric sucrose solution was 14% (v/v) in tap water. The blood alcohol concentrations were determined by the alcohol dehydrogenase method provided as a diagnostic kit by Sigma Chemicals (Sigma Technical Bulletin No. 332-4V). Procedure. Animals were mated at the onset of the dark cycle and after 6 h the male was removed and the female examined for the presence of a vaginal plug. The day a plug was detected was designated day 0 of gestation and all days, both pre- and postnatal, were counted from that point. The mice were randomly assigned to ethanol treatment or control groups, although within those assigned as controls the pair-fed animals were selected on the basis of their match in body weight to alcohol-treated animals. Because of difficulties in maintaining pregnancies among the sucrose-treated dams, three of them were pair-fed with a partner receiving alcohol, even though their body weights were 20% higher. In this case food intake was adjusted so that intake per gram body weight remained equivalent. The body weight of all maternal animals was recorded on days 0, 5, 12, 19, 22, 32, and 50. On day 50 the dams were killed and the brains removed, fixed in 10% Formalin, and blotted and weighed within 1 week. All pups were counted at birth and gestation length and litter size were recorded. On day 22, pups were weighed and maternal behavior tested with four pups in the home cage. (The remaining pups were kept warm under an infrared heat lamp.) Because experiential factors during early development have been found to influence development (6), this test was done to ascertain whether or not the dam’s behavior might have been affected by
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the alcohol treatment and thereby influence the development of the pups. The behavioral test consisted of a lo-min observation period during which each dam’s activity was recorded every 5 s, including the time when the first and last pups were retrieved; an electronic beeper was used as a timer. The activities were recorded in the following 20 mutually exclusive behavioral categories. Climb-quadrupedal motion along the horizontal wire grid of the cage lid. Run-walk-quadrupedal movement on the floor of the cage, the run distinguished by its relatively higher speed. Air sniff-animal stationary and sniffing with the nose in the air, while all four legs are on the ground, or with one or both forepaws raised or leaned on. Sniff object or pup-animal stationary and sniffing at the bedding or nesting material (object) or at the pup. Nuzzle pup-animal stationary while sniffing vigorously at pup, often pushing it with the nose and pawing it with the forepaws. Carry pup/nesting material-walking or running while grasping the pup or nesting material between teeth with head slightly raised. Pull/shred paper-sitting in one place with paper in teeth and jerking it around sideways, sometimes accompanied by chewing the edges of the paper. In nest-animal is in the nest, but obscured from view by nesting material. Arrange-sitting in the nest and pushing the nesting material in all directions with forepaws. Retrieve pups-transport of pups back to the nest after the experimenter has placed them in the corner opposite the nest. Rick dig-scraping the bedding material under the abdomen with alternating movements of forepaws, and then moving it farther backward kicking both hind legs; sometimes digging with only the forepaws is seen. Push dig-pushing the bedding material in front with alternating movements of forepaws accompanied by running. Push dig usually occurs after a sequence of kick dig, with the result that the material removed while digging a hollow is piled to one side of the cage. Groom-attending to any part of its own body, by wiping with the forepaws (usually the head), licking, scratching with the hind paws, or nibbling. Eat-chewing and swallowing pieces from a food pellet, which may be held between the forepaws. Drink-swallowing water obtained by licking at the spout of the drinking bottle.
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Although all behavioral testing was carried out by the same experimenter who administered the treatment, precautions, such as obscuring the identification tags, were taken to preserve objectivity. Because the CC in this strain attains the adult range of size about 1 week after birth (15), by measuring the structure on day 26 the possibility of a ceiling effect was minimized. Therefore, on day 26 all pups were weighed and sexed, and half the litter from each sex was chosen randomly, anesthetized with chloroform, and perfused intracardially with physiologic saline followed by 10% buffered Formalin. The brains were exposed by removal of the skull cap and suspended 24 h in 10% Formalin before being removed. They were trimmed by cutting vertically through the brain stem at the base of the cerebellum, blotted, and weighed. The brains were then embedded in gelatin, sectioned in the sag&al plane at 33 pm, and stained with hemotoxylin and eosin. The cross-sectional areas of the CC and CA were determined from tracings of midsagittal sections as described elsewhere (17). All viewing was done without prior knowledge of the experimental status of the tissue. Starting on the morning of day 26, the remaining animals were gradually weaned off alcohol until day 3 1, when they were receiving only water. On day 32 the pups were weighed and their behavioral development tested as follows: Righting reflex-does S return rapidly to its feet when placed on its back? Cliff aversion-does S withdraw from the edge of a flat surface when its snout and forepaws are placed over the precipice? Forelimb and hind limb grasp reflex-does S grasp strongly the barrel of an 18-gauge needle when it is touched to the palm of each forepaw (hind paw)? Vibrissa placing reflex-does S place its forepaw onto a cotton swab stroked across its vibrissae? Level screen test-can S hold onto a piece of 288-mesh aluminum screen when S is dragged across it horizontally by the tail? Vertical screen test-can S hold onto the screen when this is placed vertically? Screen climbing test-can S climb the vertical screen using both foreand hind limbs? Pole grasp-can S grasp the shaft (2.5 mm) of a cotton swab firmly with both fore- and hind paws? Forelimb and hind limb stick grasp-can S grip firmly a 9.5-mm-wide wooden stick with forepaws (hind paws)? Eyes open-are both eyes fully open? Visual placing reflex-does S extend its forelimbs when its is lowered rapidly toward a flat surface?
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Auditory startle response-does S show a whole-body startle response when a loud snap of fingers occurs less than 15 cm away? Each pup was assigned a score between 0.0 and 1.0 on each measure. It was possible to standardize the composite score of the animal in terms of behavioral age by substituting it into the regression equation y = 24.40 + 11.14x - 1.092 where y = age and x = score. This equation was derived by testing separate litters of a standard B6D2FI/B6D2FI hybrid cross on the behavioral test battery on each of gestation days 27 through 36 and reflects the relationship in this population between chronologic age and the litter mean behavioral scores (14). Using this scale, inbred animals were shown to be retarded in behavioral development, as were animals that were malnourished during gestation (16, 18). Litters were monitored daily and the day recorded when all animals in a litter had open eyes. On day 50 the dams and pups were weighed and their body lengths measured. They were then anesthetized with an overdose of sodium pentobarbital and perfused with physiologic saline and 10% buffered Formalin. The brains were removed and weighed as described above. Frozen sections of the brain were stained with metachromatic thionin and the CC and CA measured as described. Blood alcohol concentrations were measured on a separate but comparable group of pregnant animals between days 14 and 18 of gestation between 1400 and 1700 h. Blood was removed from the heart into a heparinized vial and stored for 24 to 48 h prior to analysis. Analysis. The data were analyzed using the GLM (general linear model) provided by SAS (Statistical Analysis System) for analysis of variance. Because of the hierarchical nature of the design, i.e., litters nested within treatment groups, the between-litter variance was used as the error term. Groups means were compared by preplanned t tests; the significance level was set at (r = 0.05. RESULTS Pup Variables. The data on the variables related to the pups are presented
in Table 1. Body weight. On each of days 22, 26, 32, and 50, the pups of the untreated control group had the highest body weight, and although the ethanol-treated pups weighed less than these, the differences were not statistically significant. Surprisingly, the pups of the pair-fed control group weighed less than either of the other two groups and were significantly lighter than the pups of the untreated control group. The same results held true of body length on day 50.
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TABLE 1 Effect of Ethanol Consumed from Day 5 to Day 26 Postconception on Pup Variables in BALB/c Mice0 Ad libitum-fed
Days postconception MY
wwt
Alcohol-fed (7)
Pair-fed sucrose (7)
lab chow
(6)
(9)
22 26 32 50
2.01 3.87 7.80 15.64
k 0.03 kO.15 + 0.20 + 0.31
1.89 3.36 6.99 14.89
+ + f k
0.04* 0.15* 0.19* 0.40”
2.15 4.10 8.20 16.31
+ 0.11*
8.26
+ + f +
0.04’ 0.13c 0.20’ 0.32’
Body length (cm) 50
7.96
It: 0.07
7.79
Brain weight (g) 26 50
0.2548 2 0.005” 0.4901 f 0.004*
0.2766 + 0.007 0.5012 + 0.006
0.2801 f 0X107~ 0.5104 + 0.003’
Corpus callosum length (mm) 26 50
2.321 k 0.176 3.368 zk 0.055<
2.048 -t 0.175 3.032 + 0.177”
2.266 + 0.164 3.412 + 0.093’
Corpus callosum ares (mm 2, 26 50
0.7786 + 0.059 1.0259 * 0.017
0.6665 k 0.056 0.9194 + 0.052*
0.7998 + 0.056 1.0914 + 0.028’
Anterior commissure (mm 2, 26 50
0.1109 & 0.003 0.1368 f 0.006*
0.1110 + 0.003 0.1551 * 0.006
0.1199 + 0.005 0.1576 + 0.005’
Behavioral scored 32
0.62
+ 0.02
0.63
+ 0.02
0.62
f 0.02
Eye opening (days)
35.00
f 0.446
34.29
f 0.29
33.50
+ 0.22E
+ 0.07c
’ Data are presented as f & SE. Parentheses show number of litters. ** Means with different superscripts in the same row are significantly different, P < 0.05. d Behavioral age Q = 30.9 days. CalcuIated by substituting behavioral score (x) into equation y = 24.40 + 11.14-x - 1.09x2.
Bruin weight. On both days 26 and 50, in pups of the ethanol-treated group the brains weighed significantly less than those of the untreated control group (one-tailed test) whereas the pair-fed control group did not differ significantly from either group. These results do not covary with those on body weight, suggesting that brain growth was affected independently of differences in body weight between the groups.
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Corpus callosum. On day 26 there were no significant differences among the groups in the area of the CC, but on day 50 this variable was significantly smaller in the pair-fed control group than in the untreated control group. The alcohol-treated group was not significantly different from either of the control groups. A similar pattern of results was seen for CC length, except on day 50 when the pair-fed control differed from both other groups. Only two pups in the study were found to be acallosal at day 50 and these originated from one pair-fed control litter; they were not included in further analysis. The effects on the corpus callosum do not reflect the same betweengroup differences seen in brain weight, where the brains of the ethanol groups weighed the least, indicating that the effect in the pair-fed control groups was independent of an effect on overall brain growth. This is supported by the fact that on both days 26 and 50 there was no significant linear correlation between brain weight and CC area in the untreated control group (day 26, r2 = 0.0034; day 50, r2 = 0.0032; P > 0.05). Anterior commissure. Again there were no significant differences among the groups on day 26, but on day 50 the mean area of the CA in pups of the alcohol-treated group was significantly smaller than that of the untreated control group. This effect appeared to be related to the reduced brain weight in these animals inasmuch as the ratio of CA area to brain weight was not significantly different among the groups. Behavioral score. There was no indication of behavioral retardation due either to inadequate nutrition or the consumption of ethanol. All groups had a behavioral age of 30.9 days, which is within the range of scores expected of inbred animals (16). However, pups of dams treated with ethanol opened their eyes an average of 1.5 days later than those of the untreated control group. Maternal Variables. The data on the variables related to the dams are presented in Table 2. Food and fluid consumption. Caloric intake did not differ between the groups of animals that consumed ethanol and the untreated control group. During the 3rd week, intake of the pair-fed control group was lower than the ad libitum-fed group, reflecting the fact that on some occasions the mice did not eat their entire ration of food. However, fluid intake during the last 2 weeks of treatment was significantly lower in the experimental group, which consumed an average of 20% of their calories as ethanol in the 1st week of treatment, decreasing to 18% during the next 2 weeks. This is equivalent to 16 g/kg ethanol per day, and should be considered in relation to a metabolic rate of alcohol elimination of 550 mg/kg/h in the mouse, compared with 300 mg/kg/h in rat and 100 mg/kg/h in man (21). This may partially explain the low blood alcohol concentrations we observed (4.58 f 0.64 mg/lOO ml, N = 6).
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TABLE 2 Effect of Ethanol Consumption from Day 5 to Day 26 Postconception on Maternal Variables in BALB/c Mice’ Ad libitum-fed
Days postconception Body weight (g) 5 12 19 22 26 32 50
Alcohol-fed (7) 20.71 23.54 34.17 24.27 24.64 25.89 24.37
+ f f f f + f
0.43’ 0.16 1.14 0.31 0.49’ 0.44’ 0.39’
Pair-fed sucrose (7) 22.74 23.70 32.53 23.10 23.13 25.17 26.51
+ k f f + + +
0.53’ 0.52 1.80 0.62* 0.55* 1.03’ 0.58’
lab chow
(6) 21.33 24.62 35.28 25.52 27.50 28.33 25.83
+ + k + + + +
0.51 0.69 1.51 0.75’ 0.68’ 0.76’ 0.33’
Brain weight (g) 50
0.5023 3~ 0.012*
0.5394 + 0.010’
0.5267 + 0.004
Litter size 0 22 26
5.29 4.86 4.57
f 0.75* f 0.70* zk 0.61
7.29 6.86 5.43
f 0.36’ + 0.51C + 0.48
5.67 5.50 5.50
+ 0.61 + 0.62 f 0.62
Gestation length (days)
20.29
+ 0.18*
19.86
+ 0.14’
20.00
+ 0.00
Maternal age (days)
78.86
+ 3.92*
100.29
2 2.52’
86.67
+ 6.22
Maternal behavior Locomote Air sniff Drink
28.57 45.57 0.14
f 2.83* * 4.57” + o.14c
18.86 24.00 9.71
f 5.14 k 2.51’ f 1.666
13.33 f 4.70c 31.67 + 6.35’ 1.67 + 1.67’
91.98 118.64 147.23
+ 4.38 f 2.58 * 6.94
85.88 110.86 132.48
f 1.59 _t 2.10 + 5.49”
85.68 122.00 168.02
k 4.88 _t 7.17 + 11.19’
Fluid intake (ml) 5-11 12-18 19-26
34.23 40.31 48.74
f 1.42 + 1.736 f 3.90*
37.89 45.19 56.83
f 2.39 zk 3.94 + 6.17
39.53 52.37 71.97
+ 1.29 f 2.23’ + 4.52’
% Ethanol-derived calories 5-11 12-18 19-26
20.55 18.71 18.13
Z!T0.50 k 0.79 f 0.89
4.58
+ 0.64
Caloric intake (kcal) 5-11 12-18 19-26
Blood alcohol wncentration (mg/lOO ml)
o Data are presented as X + SE. Parentheses show number of litters. bSMeans with different superscripts in the same row are significantly different, P < 0.05.
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Maternal age. The dams in the pair-fed control group were an average 22 days older than the ethanol-treated group, a statistically significant difference in age. The reason for this difference was that many animals initially assigned to the group fed sucrose failed to maintain their pregnancies, and therefore had to be replaced later. The untreated control group did not differ in average age from either of the other two groups. Body weight. At the beginning of treatment on day 5, the dams in the pair-fed control group weighed significantly more than those in the ethanoltreated group, although neither differed from the untreated control group. This can be explained by the age differences reported above. There were no differences at days 12 and 19 during gestation, but on day 22 the animals in the pair-fed control weighed significantly less than those in the untreated control group; on days 26 and 32 both the pair-fed control and the ethanoltreated animals weighed significantly less than this control group and on day 50 the ethanol-treated group weighed significantly less than either control group. Brain weight. The brains of the dams that had consumed ethanol weighed significantly less than those of the pair-fed control group. Litter size. At birth and day 22, the ethanol-treated dams had significantly smaller litters than those of the pair-fed control group; by day 26 this difference was no longer apparent due to the fact that many sucrose-fed pups died during that period. Gestation length. The ethanol treatment led to a gestation period almost 0.5 days longer than that in the pair-fed control group. Maternal behavior. The behavioral categories of climbing, running, and walking were combined into a single “locomotion” variable, and the alcohol-treated dams had significantly higher scores than the untreated control group. Air sniffing also increased significantly in the alcohol group compared with both control groups. The animals in the pair-fed control group had significantly higher drinking scores than those in the other two groups, a reflection of the pair-feeding procedure in which the sucrose-fed animals consumed all their rations within a short time after being fed. DISCUSSION The results of this study showed that exposure to ethanol from days 5 to 26 postconception caused a decrease in average brain weight on day 26 and a decrease in both average brain weight and average CA area on day 50. The effect on the area of the CA was, however, not independent of the effect on brain weight. Ethanol caused neither an increase in the number of acallosal animals nor an effect on callosal growth. This finding is in
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contrast with the results of the earlier study (19) in which a similar regimen of ethanol exposure resulted in a smaller cross-sectional area of the CC on day 19 postconception. One possible explanation for this difference might be that the two studies used two different sublines of BALB/c mice; those in the first study were BALB/cCF whereas the present study used BALB/ cCRBL. Another difference is that in the first study the alcohol intake ranged between 20 and 22% of the dietary calories, whereas in the present study it was between 18 and 20%. It is doubtful, however, that this small difference could account for the discrepancy in outcome. An alternative explanation might be that when the CC is growing rapidly as on day 19, a small developmental delay will cause a significant difference in size, but by day 26, when growth will have slowed down, the effects will not be as dramatic. It should be noted that five brains, all from alcohol-treated animals, disintegrated before they could be examined histologically, which may lend a conservative bias to the results. It appears that the ethanol treatment also affected the adult brains; 3 weeks after cessation of treatment these dams had significantly smaller brain weights than the sucrose-treated controls. This may be further evidence for neuropathology caused by chronic intake of ethanol (8). What is interesting about our results is the unexpected effect on the growth of the offspring in the pair-fed sucrose group. Body weight was significantly smaller in this group than in the untreated control group at all ages, and although there was no effect on brain weight, the cross-sectional area of the CC was significantly reduced at day 50. One female pup in this group showed anomalous development of the pelvis and tail; this is of interest because sacral-coccygeal defects have been reported to a much higher degree in the offspring of diabetic women (11). Similarly, maternal weight on day 22 showed that this group weighed the least, suggesting that the animals may have been unable to utilize the calories provided as sucrose. It has been shown that hyperahmentation with hypertonic glucose solutions may interfere with insulin regulation and thereby engender hyperglycemia (3). A recent study (2) found that the offspring of diabetic rats were markedly smaller in birth weight than their controls; raising questions about whether or not sucrose is the appropriate control for the empty calories provided by ethanol; certainly our results suggest that a high intake of sucrose may itself be deleterious to growth. In this respect it is interesting to note recent data that also suggest deleterious effects of a sucrose diet on reproduction and survival in rodents (9). The dosage of alcohol used in this study did not affect the behavioral development of the pups, although eye-opening was delayed, which has been observed in previous studies in rats ( 10) with a trend being evident in
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mice (7). The ethanol-treated mothers showed increased activity, which replicates previous findings indicating that small doses of ethanol cause an increase in locomotor and exploratory activity (20). In summary, the results of this study indicated that moderate ethanol consumption from days 5 to 26 postconception resulted in smaller brains measured at day 50 postconception. The area of the CA was also smaller, but only in relation to the smaller brain weight. Contrary to expectation, there was no observable effect of ethanol on the growth of the CC although there did appear to be an adverse effect due to the sucrose treatment. These findings do not support the hypothesis that moderate ethanol treatment has an enduring effect on the growth of these two forebrain fiber tracts. REFERENCES I. ABEL, E. L. (Ed.). 1984. Fetal Alcohol Syndrome and Fetal Alcohol Effects Plenum, New York. 2. AERTS, L., AND F. A. VAN ASSCHE, 198 1. Endocrine pancreas in the offspring of rats with experimentally induced diabetes. J. Endocrinol. 88: 8 l-88. 3. BERDANIER, C. D. AND M. V. KAMINSKI. 1976. Carbohydrate nutrition and hyperalimentation. Pages 129-145 in C. D. BERDANIER, Ed., Advances in Modern Nutrition, Vol. 1. Wiley, New York. 4. CHERNOFF, G. 1977. The fetal alcohol syndrome in mice: an animal model. Teratology 15: 223-230.
5. CLARREN, S. K., E. C. ALVORD, JR., S. M. SUMI, A. P. STREISGUTH, AND D. W. SMITH. 1978. Brain malformations related to prenatal exposure to ethanol. J. Pediatr. 92: 6467. 6.
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CRNIC, L. B. 1983. Effects of nutrition and environment on brain biochemistry and behavior. Dev. Psychobiol. 16: 129-145. EWART, F. G., AND M. G. CUTLER, 1979. Effects of ethyl alcohol on development and social behavior in the offspring of laboratory mice. Psychopharmacology 62: 247-25 1. LIEBER, C. S. (Ed.). 1982. Medical Disorders of Alcoholism: Pathogenesis and Treatment. Saunders, Philadelphia. SHAFRIR, E., AND J. H. ADLER. 1984. Effect of long-term sucrose diet on the reproduction and survival of spiny mice (Acornys cahirinus). Nut. Res. 4, 495-501. SHAYWITZ, B. A., G. G. GRIFFIETH, AND J. B. WARSHAW. 1979. Hyperactivity and cognitive deficits in developing rat pups born to alcoholic mothers: an experimental model of the expanded fetal alcohol syndrome (EFAS) Neurobehav. Toxicol. 1: 113122. SOLER, N. G., C. H. WALSH, AND J. M. MALINS. 1976. Congenital malformations in infants of diabetic mothers. Q. J. Med. 178, 303-313. WAHLSTEN, D. 1974. Heritable aspects of anomalous myelinated fiber tracts in the forebrain of the laboratory mouse. Brain Res. 68, l-18. WAHLSTEN, D. 1974. A developmental time scale for postnatal changes in brain and behavior of B6D2F, mice. Brain Res. 72: 25 l-264. WAHLSTEN, D. 1975. Genetic variation in the development of mouse brain and behavior: evidence from the middle postnatal period. Dev. Psycho&o/. 8: 371-380. WAHLSTEN, D. 1984. Growth of the mouse corpus callosum. Dev. Brain. Res. 15: 59-67.
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