Effects of prenatal ethanol exposure on ethanol-induced locomotor activity in rats

Effects of prenatal ethanol exposure on ethanol-induced locomotor activity in rats

Alcohol. Vol. 6, pp. 353-356. © Pergamon Press plc. 1989. Printed in the U.S.A. 0741-8329/89 $3.00 + .00 Effects of Prenatal Ethanol Exposure on Eth...

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Alcohol. Vol. 6, pp. 353-356. © Pergamon Press plc. 1989. Printed in the U.S.A.

0741-8329/89 $3.00 + .00

Effects of Prenatal Ethanol Exposure on Ethanol-Induced Locomotor Activity in Rats G A R Y E. R O C K M A N , 2 L Y N N E L I Z A B E T H M A R K E R T A N D M I C H E L E D E L R I Z Z O

Department of Psychology, University of Winnipeg 515 Portage Ave., Winnipeg, Manitoba, Canada R e c e i v e d 10 A u g u s t 1988; A c c e p t e d 11 M a y 1989

ROCKMAN, G. E., L. E. MARKERT AND M. DELRIZZO. Effects of prenatal ethanol exposure on ethanol-induced locomotor activity in rats. ALCOHOL 6(5) 353-356, 1989.--In adulthood, offspring with Fetal Alcohol Effects exhibit different responses to ethanol than nonexposed offspring; however, the majority of this research has utilized high levels of ethanol prenatally with resultant behavioural and/or physical anomalies. The present research focused on moderate prenatal ethanol exposure and subsequent challenge with low doses of ethanol. Following impregnation, female rats were exposed to between 3--4 g/kg of ethanol in a saccharin solution daily in a voluntaryfree-choice with water during one of each trimester or during all trimesters. There was a no-ethanol control group which received saccharin only as well as a group of foster dams who drank only water. At 30 days of age, male offspring were tested in an open-field after injection of either 1 g/kg ethanol or a comparable volume of saline. Rats exposed prenatally to ethanol in the second trimester showed enhanced locomotor activity. However, rats exposed prenatally to ethanol in either the first, third or during all trimesters showed no significant augmentation in activity. The results are discussed in terms of the adverse effects of moderate levels of prenatal ethanol exposure on fetal neurochemical development, specifically, the excitatory and inhibitory systems. Prenatal

Ethanol

Open field

CONSIDERABLE research has been done investigating the effects of ethanol administered prenatally. These studies have yielded interesting yet incomplete information. One reason for the lack of completeness in previous animal studies of ethanol exposure early in life concerns the frequent use of high doses of ethanol which have yielded data concerning some of the adverse consequences of such ethanol administration. For example, it has been shown that high doses of ethanol administered prenatally can alter the nutritional characteristics of the intrauterine environment (17), size, body weights, cerebellar development, (17,18), openfield behavior (6,10) and general activity levels of affected offspring (7,9). In addition, there is evidence to suggest that high levels of prenatal ethanol exposure may result in an increased preference for ethanol (6,21). Some support has been given to the notion that high levels of prenatal ethanol exposure cause changes in brain neurochemical activity. Although the subject has a controversial literature (13), adverse effects of ethanol on the development of offspring CNS, including altered brain norepinephrine (NE) and 5-hydmxytryptamine (5-HT) activity (12, 15, 16) and a reduction in sensitivity to NE of the [3-adrenoceptor coupled adenylate cyclase system (23) have been reported. In addition, inhalation exposure to ethanol has recently been shown to cause significant changes in levels of NE only in paternally-exposed offspring whereas 5-HT levels were

reduced in offspring from both paternally- and maternally-exposed rats (19). However, existing research evidence has failed to consistently specify any significant effect of low to moderate doses of prenatal ethanol exposure. For example, when animals were exposed to ethanol prenatally, there was no evidence of gross body malformations, deficiencies in learning and, in general, no teratogenic behavioral effects (1-4). However, other researchers examining the effects of low doses of ethanol on central NE functioning in adult rats, have suggested the possibility that low to moderate levels of prenatal ethanol exposure may influence the development and function of fetal central NE processes (25). In addition, it has been demonstrated that female offspring of dams intubated with ethanol exhibited tolerance to several drugs including alcohol (5). Recent data from this laboratory suggest that prenatal exposure to low to moderate levels of ethanol (that of a typical "moderate drinker") may induce central neurochemical alterations that are salient enough to be detected in adulthood (11). Specifically, in this latter study, animals were prenatally exposed to 2-3 g/kg of ethanol daily followed by exposure to a voluntary free-choice between ethanol and water. Once ethanol drinking was established, animals' ethanol drinking was "challenged" with 5-HT uptake inhibition. It was observed that ethanol exposure in the first trimester led to greater sensitivity to the effects of zimelidine in attenuating ethanol consumption (11). In addition, it was sug-

~Supported by the Manitoba Mental Health Research Foundation. 2Requests for reprints should be addressed to Gary E. Rockman.

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gested that this sensitivity was related to the effects of prenatal ethanol exposure on NE and/or 5-HT systems. Finally, it is important to emphasize that this greater sensitivity to zimelidine occurred in animals without obvious physical defects, suggesting that the teratogenic effects are subtle. Consequently, it is possible that the resulting abnormalities from low to moderate levels of prenatal ethanol exposure may be covert until the organism is in a situation which would cause the "weak link'" of the system to show itself. This may occur as a result of various "'challenges" in adulthood as noted in the research described previously [e.g., stress challenge, drug challenge, ethanol challenge (5, 6, 1 l, 26)1. The present study was done in an effort to determine the effects of moderate doses of prenatal ethanol on subsequent response to ethanol in an open field. Rats exposed to ethanol prenatally should exhibit a differential response to ethanol as adults than nonexposed controls. The effects of moderate doses of ethanol, which reflect the typical social drinker, have not been studied extensively. In addition, to approximate a more "'normal" situation, the present author has recently developed a voluntal y fiee-choice method of prenatal ethanol exposure ( 11 ) which reduces and/or eliminates the inherent difficulties of forced-choice methods. In the present study, rats which had been prenatally exposed to moderate doses of ethanol (3-4 g/kg) were injected at 30 days of age with either ethanol ( 1.0 g/kg) or saline before behavioural testing. Open-field behavioural measures included: step-down latency, ambulation, rearing, and number of boli. METHOD

Subiect~ Forty male and 40 female Holtzman rats were used to breed the offspring of which 70 males (10 groups, n = 7 ) were used in the experiment. The females and males were housed at a ratio of 1:1 for purposes of impregnation. The first day of pregnancy was determined by the presence of a sperm plug, after which females were place in a standard cage housing and randomly assigned to the various experimental and control groups.

Procedure Prenatal ethanol exposure. All of the dams in the five experimental groups were exposed to 0.4% saccharin/water (w/v) solution throughout the entire gestation. However, the groups differed in the timing of ethanol exposure, Groups were exposed to ethanol during either the first trimester (days 1-7), the second trimester (days 8-14), the third trimester (days 14-21 ), or throughout the entire gestational period (Days 1-21). The Control group was exposed to saccharin/water (0.4%), they were not exposed to ethanol prenatally. A final group of females was used as crossfoster dams and received water only throughout gestation. The ethanol/saccharin mixture and the saccharin only mixture were presented by free-choice between the solution and water, rather than by forced-choice or intubation (11). The procedure for the free-choice method was as follows: Animals were presented with two tubes, one containing water, and the other containing either the ethanol solution (4% v/v in 0.4% Saccharin w/v) or the 0.4 (w/v) Saccharin solution only. The side of presentation of the solutions was altered daily to eliminate the possibility of formation of a position preference. The dams were weighed daily and presented with between 30 to 55 milliliters of ethanol/saccharin solution to result in a daily level of consumption between 3M- g/kg of ethanol, or a comparable volume of Saccharin only solution. Females not consuming sufficient levels of either solution were excluded from the study. It is important to note that this method of prenatal ethanol exposure does not interfere with food and water consumption (11). Thus, the confounding effects of malnutrition

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FIG, 1. Mean ambulation of rats exposed to prenatal ethanol (n = 7 per group) during 1st trimester (lst), second trimester (2nd), third trimester (3rd), all trimesters (all) and controls, subsequently injected with ethanol IETOH) or saline (Sal). and dehydration would be reduced or eliminated (8). As well, this method is more reflective of moderate, periodic ethanol consumption in humans. Standard lab chow was available ad lib and all the dams were handled daily. Near the date of parturition, false bottoms and shavings were placed in the single cages. Postnatal treatment~offspring. Within 24 hours of parturition, litters were culled of female pups and male pups were placed in nesting boxes with cross-foster females who had given birth in the previous 24 hours. Cross-fostering was used to prevent possible behavioural defects of the mother affecting the offspring. The dams had free access to food and water and were left undisturbed except for purposes of cleaning the nesting boxes. At 21 days of age. offspring were removed and placed in individual cages with food and water ad lib at room temperature 25 ° C, 12 hour light/dark cycle; lights on at 0700 Hours. All animals were handled every other day. ()pen field testing. Offspring were tested in a metal, circular open field measuring 191.41 cm in circumference. The field was painted flat black with thirty 10.16 × 10.16 cm squares marked off in white. In the centre of the field was a ceramic platform with a circumference of 50 cm, measuring 3.81 cm in height. When the rats were 30 days of age, testing began. Testing took place between 0900 and 1500 Hours daily under normal room lighting. Subjects were tested daily for three consecutive days. Half of each group of offspring was weighed and injected (IP) with 1.0 g/kg of ethanol (20% v/v) in saline solution. The remaining animals from each of the groups were weighed and injected with a comparable volume of saline. One minute after injection of either ethanol or saline, each rat was placed in the centre of the platform in the open field and timed for 3 minutes, The dose of ethanol and interval between injections and testing was established by pilot studies is similar to the methodology described by Spivak et al. (24). The open-field testing was videotaped for later scoring. Measurements were: step-down latency (time in seconds to leave the platform on which the rat was placed), ambulation (the numbers of squares entered) rearing and number of boli. Number of boli were counted at the time of testing. The open field was cleaned after each subject was removed. RESULTS

Results were analyzed by two-way analysis of variance ol

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Group (lst, 2nd, 3rd, or All Trimesters, Control) x Treatment (Ethanol or Saline Injection). This analysis was performed on an aggregation of scores over the three-day testing period, a method considered more accurate than analysis of single day scores (20). The dependent variable of augmentation of ambulation as measured by the number of squares entered in the open field subsequent to either an ethanol or saline challenge showed a significant main effect of group, F(4,60)= 2.51, p<0.05, and a main effect of treatment, F(1,60)= 41.42, p<0.01. Most importantly, there was a group × treatment interaction, F(4,60) = 2.53, p<0.05. Figure 1 illustrates the pattern of results of ambulation in the open field after challenge with either ethanol or saline. The main finding as revealed by post hoc Tukey tests was that rats exposed to ethanol in the second trimester and subsequently challenged with ethanol showed significant augmentation of ambulation in the open field as compared with their own saline injected group (p<0.01) as well as the control group injected with ethanol (p<0.05). Further post hoc Tukey tests showed that exposure to ethanol during the first trimester and subsequent ethanol challenge resulted in no augmentation of activity as compared with first trimester/saline challenged group or with either of the control groups. The group exposed to ethanol in the third trimester and subsequently challenged with ethanol showed augmentation of activity compared to the third trimester/saline challenged group (p<0.05). However, this group was not significantly different from the overall control group. Locomotor activity of rats which received ethanol throughout all trimesters did not differ significantly from their own saline-challenged group or the control groups. Similarly, control rats (the group which was not exposed to ethanol prenatally) showed small increases in locomotor activity following ethanol injection, however, this increase in locomotor activity only approached significance. No significant main effects or interactions were observed for the step-down latency, rearing or number of boli measures. Consequently, these data are not presented. Finally, no obvious physical defects or significant differences in weight were noted in either the dams or offspring. DISCUSSION The results showed that exposure to moderate levels of ethanol in the second trimester resulted in augmented ambulation when subjects were challenged with ethanol prior to open-field placement. Further, some augmentation was evident in the "third trimester" group but this group was not significantly different from the overall control group. The "all trimesters" group showed no augmentation and it is worth noting that this is with the addition of first trimester exposure which by itself had no effect on ambulation. Overall, the results indicate that prenatal ethanol exposure causes behaviour differences when exposure takes place during the second trimester. This is consistent with a recent report

indicating that prenatal exposure to moderate levels of ethanol (4 g/kg) in the second trimester alters neuronal development (28). Perhaps the results of the present study may be explained by the timing of prenatal ethanol exposure and the corresponding development of the fetal brain. Although specific neurotransmitters may not be present in the brain at the time of ethanol administration (14), the precursor chemicals are present. Previous research has shown that excitatory systems develop before inhibitory systems (8,14) and the results of the present study may reflect this. Perhaps the lack of augmentation of locomotion in the first trimester resulted from defects in development of the excitatory system, i.e., ethanol injections would normally cause an increase in ambulation in the open field but this did not occur in the 1st trimester group, which was not significantly different from its own control group. This is consistent with the previous findings in this laboratory (11) which revealed an effect of prenatal ethanol in the first trimester, i.e., rats exposed to moderate levels of ethanol in the first trimester were more sensitive to the effects of zimelidine in decreasing ethanol consumption than any other group. Thus, the present study is consistent with previous results suggesting an adverse effect of prenatal ethanol exposure on excitatory mechanisms. As well, the significant increase in ambulation of the group exposed to ethanol in the second trimester and subsequently challenged with ethanol would be consistent with defects in the development of the inhibitory system (22). If this system develops after the excitatory system, it would follow that increased ambulation would result from challenge with ethanol due to lack of appropriate functioning of inhibitory mechanisms. Other studies utilizing higher levels of ethanol have also resulted in findings of increased or decreased levels of NE and/or 5-HT (12, 15, 27). As the inhibitory systems continue to develop (27), the adverse effects of prenatal ethanol during the third trimester are less evident. This is demonstrated in the lesser augmentation of locomotion in the 3rd trimester group after injection with ethanol in comparison with the 3rd trimester saline-injected group. It is not apparent why exposure to ethanol in all trimesters did not result in significantly adverse effects, although this pattern of findings was also evident in previous studies (11). A possible explanation is that both the excitatory and inhibitory systems work to reestablish a balance between inhibition and excitation with some recovery although never returning to normal levels. In general, however, the effects of prenatal ethanol exposure as demonstrated by behavioural measures seemed to reflect the timing of exposure and stage of development (8). In conclusion, the present study revealed some results which, taken together with past research, provide evidence that prenatal exposure to moderate doses of ethanol resulted in behavioural alterations after subsequent "challenge" with ethanol. Thus, exposure of rats and human infants to moderate levels of ethanol in utero may account for altered subsequent responses to ethanol in adulthood.

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ethanol. Neurotoxicol. Teratol. 10:15-22; 1988. 20. Ossenkopp, K. Macrae, L. K.; Teskey, G. C. Automated multivariate measurement of spontaneous motor activity in mice: Time course and reliabilities of the behavioral measures. Pharmacot. Biochem. Behav. 27:565-568; 1987. 21. Randall, C. L.; Hughes, S. L4 Williams, C. K.; Anton, R. F. Effect of prenatal alcohol exposure on comsumption of alcohol and alcohol induced sleep time in mice. Pharmacol. Biochem. Behav. 18:325329; 1983. 22. Riley, E. P.; Lochry, E. A.; Shapiro, N. R.; Baldwin, J. Response perseveration in rats exposed to alcohol prenatally. PharmacoL Biochem. Behav. 10:255-259; 1979. 23. Salinas, M.; Fernandez, T. Effects of chronic ingestion of alcohol in the pregnant rat on catecbolamine sensitive adenylate cyclase in the brain of mothers and their offspring. Neuropharmacology 22:12831288; 1983. 24. Spivak, K.; Aragon, C. M. G.; Amit, Z. Alterations in brain aldehyde dehydrogenase activity modify the locomotor effects produced by ethanol in rats. Alcohol Drug Res. 7:481-491; 1987. 25. Strahlendoff, H. K.; Strahlendorf, J. C. Ethanol suppression of locus coerulus neurons: Relevancy to the fetal alcohol syndrome. Neurobehav. Toxicol. Teratol. 5:221-224; 1983. 26. Taylor, A. N.; Nelson, L. R.; Branch, B. J.; Kokka, N.; Poland, R. E. Altered stress responsiveness in adult rats exposed to ethanol in utero: neuroendocrine mechanisms. In: Mechanisms of Alcohol Damage in Utero. Ciba Foundation Symposium 105. London: Pitman; 1984:47-65. 27. Volk, B. Neurohistological and neurobiological aspects of fetal alcohol syndrome in the rat. In: Yanai, J., ed. Neurobehavioral teratology. New York: Elsevier Science Publishers BV; 1984:163193. 28. Vorhees, C. V.; Rauch, S.; Hitzemann, R. Effects of short-term prenatal alcohol exposure on neuronal membrane oreder in rats. Dev. Brain Res. 38:161-166: 1988.