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Behavioral changes in rat following perinatal exposure to ethanol
Neuroscience Letters, 47 (1984) 145-148 Elsevier Scientific Publishers Ireland Ltd.
145
NSL 02735 B E H A V I O R A L C H A N G E S IN R A T F O L L O W I N G P E R I N A T A L E X P O S U R E TO ETHANOL
KANHAIYA R. SHAH and MICHAEL WEST Department of Physiology, 770 Bannatyne Avenue, University of Manitoba, Winnipeg, Manitoba R3E OW3 (Canada)
(Received December 29th, 1983; Revised version received and accepted March 14th, 1984)
Key words: perinatal ethanol exposure - locomotion - standing - nose poking
Behavioral changes followingpre- and postnatal ethanol administration were examined in rat pups of various ages. A significant increase in locomotoractivityoccurredat all ages (16, 22 and 30 days) examined. Nose poking activity in 22-day-old pups and standing activity in 30-day-old pups were also significantly higher following perinatal ethanol exposure. No significant change in grooming behavior was observed. The overactivity in rat pups following perinatal ethanol exposure is consistent with the frequently observed hyperactivity in fetal alcohol syndrome children.
Since the recognition o f fetal alcohol syndrome, a distinctive pattern of abnormalities and growth deficiencies which occur in a substantial proportion of children born to alcoholic women [4,6,7,15], many studies with animal models have confirmed the teratogenic potential of ethanol exposure during gestation [2,3,13,14]. Mental retardation is the most c o m m o n feature o f ethanol teratogenicity; however, a variety of behavioral deficiencies such as hyperactivity, irritability, restlessness, fine m o t o r problems and poor finger articulation have been reported in children with fetal alcohol syndrome [5,6,9,11,12]. In animal studies, overactivity in an open field [1,2], impairment in learning a T-maze [10], deficits in shuttle avoidance performance [10] and poor performance on certain operant schedules [8] have been demonstrated following prenatal exposure to ethanol. These findings have similarities to those found in humans, and although the actions of ethanol in man cannot be isolated easily from the influence o f other factors, the usefulness of an animal model in fetal alcohol syndrome is indicated. The objective o f the present report was to develop an animal model to investigate the behavioral alterations which may result from the perinatal ethanol exposure. The effect on the locomotor, grooming, standing and nose poking activities o f rat pups at different ages following such treatment is reported. Timed pregnant Sprague-Dawley rats, housed in separate cages and maintained on a 12-h light-dark cycle, were divided into control and ethanol groups. Starting 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
146 o n d a y 8 o f g e s t a t i o n , a liquid diet c o m p o s e d o f 61°70 ( v / v ) E n s u r e (Ross L a b o r a t o i r e s ) , 6.5o70 ( v / v ) e t h a n o l a n d 32.5°7o w a t e r , was o f f e r e d to e t h a n o l g r o u p as their o n l y source o f f o o d a n d water. T h e c o n t r o l g r o u p received a similar diet except e t h a n o l was s u b s t i t u t e d with a n i s o c a l o r i c a m o u n t o f sucrose. T o ensure t h a t b o t h g r o u p s c o n s u m e d equal a m o u n t s , the c o n t r o l a n i m a l s were o f f e r e d the s a m e a m o u n t o f liquid diet t h a t was c o m p l e t e l y c o n s u m e d b y the e t h a n o l g r o u p . T h e a v e r a g e d o s e o f e t h a n o l t a k e n d a i l y was 10 g / k g b o d y weight. A t birth, each litter was culled to 8 p u p s . T h e liquid diet r e g i m e in b o t h g r o u p s c o n t i n u e d until the p u p s were 10 d a y s o l d w h e r e u p o n all m o t h e r s were o f f e r e d r e g u l a r l a b c h o w . F o u r b e h a v i o r a l p a r a m e t e r s were assessed in the p u p s at v a r i o u s ages. These were l o c o m o t i o n , g r o o m i n g ( f o r e p a w cleaning, h i n d l i m b scratching, licking), s t a n d i n g ( u p r i g h t with f r o n t p a w s a g a i n s t the wall) a n d nose p o k i n g . T h e o b s e r v a t i o n c h a m b e r was a c i r c u l a r Plexiglas enclosure 38 c m high a n d 20 c m in d i a m e t e r with small holes (1 c m in d i a m e t e r ) at the base. This was k e p t in a s o u n d a t t e n u a t e d r o o m a n d the b e h a v i o r was m o n i t o r e d via a closed circuit television. R a n d o m l y selected rats o f v a r i o u s ages were p l a c e d singly into t h e c h a m b e r a n d a l l o w e d a 5-min p e r i o d o f a c c l i m a t i o n . T h e t i m e spent in each b e h a v i o r a l c a t e g o r y was t h e n registered o n electronic s t o p w a t c h e s which were c o m b i n e d in such a m a n n e r t h a t when o n e w a t c h was switched o n the o t h e r s s t o p p e d at a n y given t i m e d u r i n g the 20-min o b s e r v a t i o n p e r i o d . S t a n d i n g a n d n o s e p o k i n g activities were m o n i t o r e d at age 30 a n d 22 days, respectively, since these activities were well p r o n o u n c e d o n these days. N o s e p o k i n g a g a i n s t t h e small holes at the b a s e o f t h e c h a m b e r was m e a s u r e d as the n u m b e r o f events t h a t o c c u r r e d d u r i n g the o b s e r v a t i o n p e r i o d . TABLE I LOCOMOTOR AND GROOMING ACTIVITIES IN RAT PUPS AFTER PRE- AND POSTNATAL EXPOSURE TO ETHANOL The animals in the experimental group received ethanol in utero and during lactation as described in the text. The control animals received an isocaloric replacement of sucrose. Values expressed are seconds (means _+ S.E.M.) spent in the activity during a 20-rain observation period. Number of animals tested for each group is shown in the parentheses. *Significantly different from control (P < 0.05, t-test). Age (days)
Locomotor activity Control
Ethanol
16 22 30
488 + 105 (10) 692 + 103 (5) 599 _+ 55(10)
742 + 100 (10)* 971 ± 82( 6)* 848 +_ 59( 8)*
Age (days)
Grooming
16 22 30
Control
Ethanol
106 + 16 (10) 252 ± 6 6 ( 6 ) 183 + 33 (10)
93 + 15 (10) 128 + 6 0 ( 6 ) 165 + 35 ( 8 )
147 There was no significant difference between the average weight gains of control and ethanol-treated mothers during the course o f study and no obvious withdrawal reactions were observed in ethanol-treated rats. The effect o f ethanol on locomotor activity is shown in Table I. The pups that were perinatally exposed to ethanol displayed a significantly higher activity than control pups at all three ages (16, 22 and 30 days old) examined. The grooming behavior, although not statistically different, was lower in ethanol pups than control at all ages observed (Table I). At age 30, the standing activity was dramatically higher in the ethanol group than in controis (ethanol: 143 + 10, n = 8; control: 43 + 9, n = 10; P < 0.05, t-test). Nose poking at 22 days also showed a significant increase in ethanol-treated pups (ethanol: 107 + 26, n = 5; control: 45 _+ 6, n = 5; P < 0.05, t-test). While there is an abundance o f literature on the effect of ethanol on the adult central nervous system and behavior, there have been only a few studies concerning the ability of early ethanol exposure (pre- and postnatal) to induce long-term neural and behavioral changes. Although causal relationships between ethanol consumption and behavior are difficult to demonstrate in man since the influence of other factors cannot be eliminated easily, important correlates may still be uncovered using controlled animal studies. In man, hyperactivity is a common feature of fetal alcohol syndrome children [5, 12]. The findings of the present report indicate that the perinatal ethanol exposure results in overactivity in rat pups. This overactivity is reflected by the increase in locomotor, standing and nose poking activities. It is unlikely that the behavioral changes observed are due to some nutritional deficiency since there was no significant change in body weights of pups between both groups and no overt evidence o f malnutrition was evident. There was no significant difference in grooming behavior between ethanol-treated and control pups (Table I). The small decrease noted in ethanol-treated pups may be a result of increase in other exploratory activities such as locomotion, standing and nose poking. The frequency of typical behavioral deficits seems higher than gross, physical malformations in fetal alcohol syndrome children. However, any effect of ethanol on mental or behavioral development is more difficult to demonstrate than the occurrence of physical anomalies. The finding of the present study is in accordance with the hyperactivity observed in fetal alcohol syndrome children. Further investigation is needed to understand the biochemical mechanisms responsible for the behavioral changes occurring after perinatal exposure to ethanol. We are grateful to the late Dr. Havlicek for his support o f this project. Also, we thank Janet Balak for typing the manuscript.
1 Bond, N.W. and DiGiusto, E.L., Effects of prenatal alcohol consumptionon open-fieldbehavior and alcohol preference in rats, Psychopharmacologia,46 (1976) 163-165.
148 2 Branchey, L. and Friedhoff, A.J., Biochemical and behavioral changes in rats exposed to ethanol in utero, Ann. N.Y. Acad. Sci., 273 (1976) 328-330. 3 Chernoff, G.F., The fetal alcohol syndrome in mice; an animal model, Teratology, 15 (1977) 223-229. 4 Clarren, S.K. and Smith, D.W., The fetal alcohol syndrome, N. Engl. J. Med., 298 (1978) 1063-1067. 5 Hanson, J.W., Jones, K.L. and Smith, D.W., Fetal alcohol syndrome: experience with 41 patients, J. Amer. Med. Ass., 235 (1976) 1458-1460. 6 Jones, K.L. and Smith, D.W., Recognition of the fetal alcohol syndrome in early infancy, Lancet, ii (1973) 999-1001. 7 Jones, K.L., Smith, D.W., Ulleland, C.N. and Streissguth, A.P., Pattern of malformation in offspring of chronic alcoholic mothers, Lancet, i (1973) 1267-1271. 8 Martin, J.C., Martin, D.C., Sigman, G. and Radow, B., Offspring survival, development, and operant performance following maternal ethanol consumption, Develop. Psychobiol., 10 (1977) 435-446. 9 Shaywitz, B.A., Fetal alcohol syndrome: an ancient problem rediscovered, Drug Ther., 8 (1978) 53-60. l0 Shaywitz, B.A., Klopper, J.H. and Gordon, J.W., A syndrome resembling minimal brain dysfunction (MBD) in rat pups born to alcoholic mothers, Pediat. Res., 10 (1976) 451-455. ll Shruygin, G.I., Characteristics of the mental development of children of alcoholic mothers, Pediatriya Mosk., 11 0974) 71-73. 12 Streissguth, A.P., Maternal alcoholism and the outcome of pregnancy. A review of fetal alcohol syndrome. In N. Greenblatt and M. Schckit (Eds.), Problems of Alcohol in Women and Children, Grune and Stratton, New York, 1976, pp. 251-274. 13 Thadani, P.V., Lau, C., Slotkin, T.A. and Schanberg, S.M., Effect of maternal ethanol ingestion on neonatal rat brain and heart ornithine decarboxylase, Biochem. Pharmacol., 26 (1977) 523-527. 14 Tze, W.J. and Lee, M., Adverse effects of maternal alcohol consumption on pregnancy and fetal growth in rats, Nature (Lond.), 257 (1975) 479-480. 15 Ulleland, C.N., The offspring of alcoholic mothers, Ann. N.Y. Acad. Sci., 197 (1972) 167-169.
145
NSL 02735 B E H A V I O R A L C H A N G E S IN R A T F O L L O W I N G P E R I N A T A L E X P O S U R E TO ETHANOL
KANHAIYA R. SHAH and MICHAEL WEST Department of Physiology, 770 Bannatyne Avenue, University of Manitoba, Winnipeg, Manitoba R3E OW3 (Canada)
(Received December 29th, 1983; Revised version received and accepted March 14th, 1984)
Key words: perinatal ethanol exposure - locomotion - standing - nose poking
Behavioral changes followingpre- and postnatal ethanol administration were examined in rat pups of various ages. A significant increase in locomotoractivityoccurredat all ages (16, 22 and 30 days) examined. Nose poking activity in 22-day-old pups and standing activity in 30-day-old pups were also significantly higher following perinatal ethanol exposure. No significant change in grooming behavior was observed. The overactivity in rat pups following perinatal ethanol exposure is consistent with the frequently observed hyperactivity in fetal alcohol syndrome children.
Since the recognition o f fetal alcohol syndrome, a distinctive pattern of abnormalities and growth deficiencies which occur in a substantial proportion of children born to alcoholic women [4,6,7,15], many studies with animal models have confirmed the teratogenic potential of ethanol exposure during gestation [2,3,13,14]. Mental retardation is the most c o m m o n feature o f ethanol teratogenicity; however, a variety of behavioral deficiencies such as hyperactivity, irritability, restlessness, fine m o t o r problems and poor finger articulation have been reported in children with fetal alcohol syndrome [5,6,9,11,12]. In animal studies, overactivity in an open field [1,2], impairment in learning a T-maze [10], deficits in shuttle avoidance performance [10] and poor performance on certain operant schedules [8] have been demonstrated following prenatal exposure to ethanol. These findings have similarities to those found in humans, and although the actions of ethanol in man cannot be isolated easily from the influence o f other factors, the usefulness of an animal model in fetal alcohol syndrome is indicated. The objective o f the present report was to develop an animal model to investigate the behavioral alterations which may result from the perinatal ethanol exposure. The effect on the locomotor, grooming, standing and nose poking activities o f rat pups at different ages following such treatment is reported. Timed pregnant Sprague-Dawley rats, housed in separate cages and maintained on a 12-h light-dark cycle, were divided into control and ethanol groups. Starting 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
146 o n d a y 8 o f g e s t a t i o n , a liquid diet c o m p o s e d o f 61°70 ( v / v ) E n s u r e (Ross L a b o r a t o i r e s ) , 6.5o70 ( v / v ) e t h a n o l a n d 32.5°7o w a t e r , was o f f e r e d to e t h a n o l g r o u p as their o n l y source o f f o o d a n d water. T h e c o n t r o l g r o u p received a similar diet except e t h a n o l was s u b s t i t u t e d with a n i s o c a l o r i c a m o u n t o f sucrose. T o ensure t h a t b o t h g r o u p s c o n s u m e d equal a m o u n t s , the c o n t r o l a n i m a l s were o f f e r e d the s a m e a m o u n t o f liquid diet t h a t was c o m p l e t e l y c o n s u m e d b y the e t h a n o l g r o u p . T h e a v e r a g e d o s e o f e t h a n o l t a k e n d a i l y was 10 g / k g b o d y weight. A t birth, each litter was culled to 8 p u p s . T h e liquid diet r e g i m e in b o t h g r o u p s c o n t i n u e d until the p u p s were 10 d a y s o l d w h e r e u p o n all m o t h e r s were o f f e r e d r e g u l a r l a b c h o w . F o u r b e h a v i o r a l p a r a m e t e r s were assessed in the p u p s at v a r i o u s ages. These were l o c o m o t i o n , g r o o m i n g ( f o r e p a w cleaning, h i n d l i m b scratching, licking), s t a n d i n g ( u p r i g h t with f r o n t p a w s a g a i n s t the wall) a n d nose p o k i n g . T h e o b s e r v a t i o n c h a m b e r was a c i r c u l a r Plexiglas enclosure 38 c m high a n d 20 c m in d i a m e t e r with small holes (1 c m in d i a m e t e r ) at the base. This was k e p t in a s o u n d a t t e n u a t e d r o o m a n d the b e h a v i o r was m o n i t o r e d via a closed circuit television. R a n d o m l y selected rats o f v a r i o u s ages were p l a c e d singly into t h e c h a m b e r a n d a l l o w e d a 5-min p e r i o d o f a c c l i m a t i o n . T h e t i m e spent in each b e h a v i o r a l c a t e g o r y was t h e n registered o n electronic s t o p w a t c h e s which were c o m b i n e d in such a m a n n e r t h a t when o n e w a t c h was switched o n the o t h e r s s t o p p e d at a n y given t i m e d u r i n g the 20-min o b s e r v a t i o n p e r i o d . S t a n d i n g a n d n o s e p o k i n g activities were m o n i t o r e d at age 30 a n d 22 days, respectively, since these activities were well p r o n o u n c e d o n these days. N o s e p o k i n g a g a i n s t t h e small holes at the b a s e o f t h e c h a m b e r was m e a s u r e d as the n u m b e r o f events t h a t o c c u r r e d d u r i n g the o b s e r v a t i o n p e r i o d . TABLE I LOCOMOTOR AND GROOMING ACTIVITIES IN RAT PUPS AFTER PRE- AND POSTNATAL EXPOSURE TO ETHANOL The animals in the experimental group received ethanol in utero and during lactation as described in the text. The control animals received an isocaloric replacement of sucrose. Values expressed are seconds (means _+ S.E.M.) spent in the activity during a 20-rain observation period. Number of animals tested for each group is shown in the parentheses. *Significantly different from control (P < 0.05, t-test). Age (days)
Locomotor activity Control
Ethanol
16 22 30
488 + 105 (10) 692 + 103 (5) 599 _+ 55(10)
742 + 100 (10)* 971 ± 82( 6)* 848 +_ 59( 8)*
Age (days)
Grooming
16 22 30
Control
Ethanol
106 + 16 (10) 252 ± 6 6 ( 6 ) 183 + 33 (10)
93 + 15 (10) 128 + 6 0 ( 6 ) 165 + 35 ( 8 )
147 There was no significant difference between the average weight gains of control and ethanol-treated mothers during the course o f study and no obvious withdrawal reactions were observed in ethanol-treated rats. The effect o f ethanol on locomotor activity is shown in Table I. The pups that were perinatally exposed to ethanol displayed a significantly higher activity than control pups at all three ages (16, 22 and 30 days old) examined. The grooming behavior, although not statistically different, was lower in ethanol pups than control at all ages observed (Table I). At age 30, the standing activity was dramatically higher in the ethanol group than in controis (ethanol: 143 + 10, n = 8; control: 43 + 9, n = 10; P < 0.05, t-test). Nose poking at 22 days also showed a significant increase in ethanol-treated pups (ethanol: 107 + 26, n = 5; control: 45 _+ 6, n = 5; P < 0.05, t-test). While there is an abundance o f literature on the effect of ethanol on the adult central nervous system and behavior, there have been only a few studies concerning the ability of early ethanol exposure (pre- and postnatal) to induce long-term neural and behavioral changes. Although causal relationships between ethanol consumption and behavior are difficult to demonstrate in man since the influence of other factors cannot be eliminated easily, important correlates may still be uncovered using controlled animal studies. In man, hyperactivity is a common feature of fetal alcohol syndrome children [5, 12]. The findings of the present report indicate that the perinatal ethanol exposure results in overactivity in rat pups. This overactivity is reflected by the increase in locomotor, standing and nose poking activities. It is unlikely that the behavioral changes observed are due to some nutritional deficiency since there was no significant change in body weights of pups between both groups and no overt evidence o f malnutrition was evident. There was no significant difference in grooming behavior between ethanol-treated and control pups (Table I). The small decrease noted in ethanol-treated pups may be a result of increase in other exploratory activities such as locomotion, standing and nose poking. The frequency of typical behavioral deficits seems higher than gross, physical malformations in fetal alcohol syndrome children. However, any effect of ethanol on mental or behavioral development is more difficult to demonstrate than the occurrence of physical anomalies. The finding of the present study is in accordance with the hyperactivity observed in fetal alcohol syndrome children. Further investigation is needed to understand the biochemical mechanisms responsible for the behavioral changes occurring after perinatal exposure to ethanol. We are grateful to the late Dr. Havlicek for his support o f this project. Also, we thank Janet Balak for typing the manuscript.
1 Bond, N.W. and DiGiusto, E.L., Effects of prenatal alcohol consumptionon open-fieldbehavior and alcohol preference in rats, Psychopharmacologia,46 (1976) 163-165.
148 2 Branchey, L. and Friedhoff, A.J., Biochemical and behavioral changes in rats exposed to ethanol in utero, Ann. N.Y. Acad. Sci., 273 (1976) 328-330. 3 Chernoff, G.F., The fetal alcohol syndrome in mice; an animal model, Teratology, 15 (1977) 223-229. 4 Clarren, S.K. and Smith, D.W., The fetal alcohol syndrome, N. Engl. J. Med., 298 (1978) 1063-1067. 5 Hanson, J.W., Jones, K.L. and Smith, D.W., Fetal alcohol syndrome: experience with 41 patients, J. Amer. Med. Ass., 235 (1976) 1458-1460. 6 Jones, K.L. and Smith, D.W., Recognition of the fetal alcohol syndrome in early infancy, Lancet, ii (1973) 999-1001. 7 Jones, K.L., Smith, D.W., Ulleland, C.N. and Streissguth, A.P., Pattern of malformation in offspring of chronic alcoholic mothers, Lancet, i (1973) 1267-1271. 8 Martin, J.C., Martin, D.C., Sigman, G. and Radow, B., Offspring survival, development, and operant performance following maternal ethanol consumption, Develop. Psychobiol., 10 (1977) 435-446. 9 Shaywitz, B.A., Fetal alcohol syndrome: an ancient problem rediscovered, Drug Ther., 8 (1978) 53-60. l0 Shaywitz, B.A., Klopper, J.H. and Gordon, J.W., A syndrome resembling minimal brain dysfunction (MBD) in rat pups born to alcoholic mothers, Pediat. Res., 10 (1976) 451-455. ll Shruygin, G.I., Characteristics of the mental development of children of alcoholic mothers, Pediatriya Mosk., 11 0974) 71-73. 12 Streissguth, A.P., Maternal alcoholism and the outcome of pregnancy. A review of fetal alcohol syndrome. In N. Greenblatt and M. Schckit (Eds.), Problems of Alcohol in Women and Children, Grune and Stratton, New York, 1976, pp. 251-274. 13 Thadani, P.V., Lau, C., Slotkin, T.A. and Schanberg, S.M., Effect of maternal ethanol ingestion on neonatal rat brain and heart ornithine decarboxylase, Biochem. Pharmacol., 26 (1977) 523-527. 14 Tze, W.J. and Lee, M., Adverse effects of maternal alcohol consumption on pregnancy and fetal growth in rats, Nature (Lond.), 257 (1975) 479-480. 15 Ulleland, C.N., The offspring of alcoholic mothers, Ann. N.Y. Acad. Sci., 197 (1972) 167-169.