Effect of prenatal ethanol exposure on response to abrupt reward reduction

Neurotoxicology and Teratology, Vol. 10, pp. 121-125. ©PergamonPress plc, 1988.Printedin the U.S.A.

0892-0362/88$3.00 + .00

Effect of Prenatal Ethanol Exposure on Response to Abrupt Reward Reduction H O W A R D C. B E C K E R , .1 C A R R I E L. R A N D A L L * A N D EDWARD P. R I L E Y t

*Medical University o f South Carolina and Veterans Administration Medical Center, Charleston, S C and t S t a t e University o f N e w York at Albany, Albany, N Y R e c e i v e d 18 J u n e 1987 BECKER, H. C., C. L. RANDALL AND E. P. RILEY. Effect of prenatal ethanol exposure on response to abrupt reward reduction. NEUROTOXICOL TERATOL 10(2) 121-125, 1988.--The purpose of this study was to examine the effects of prenatal ethanol exposure on an appetitively-motivated behavioral task (consummatory negative contrast) that involves quantification of an animal's response to an abrupt, unexpected reduction in reward (sucrose solutions). Pregnant LongEvans rats received isocaloric liquid diets containingeither 35% or 0% ethanol-derived calories on days 6--20of gestation. A pair-feeding procedure was employed, and a lab chow control group also was included. Adult male offspring from these three prenatal treatment groups were used for behavioral testing. Results indicated all groups exhibited suppressed responding subsequent to reward reduction. This effect gradually diminished in all prenatal treatment groups over several test sessions. While there was a numerical tendency for ethanol-exposed offspring to exhibit a smaller initial contrast effect (less response inhibition) and recover to control levels at a faster rate than the sucrose and lab chow control groups, this effect was not statistically significant. Thus, prenatal ethanol exposure does not appear to greatly influence the response to abrupt, partial reward reduction in adult rat offspring. Prenatal ethanol

Negative contrast

Response inhibition

OVER the past decade and a half, a great deal of clinical and basic research has established the teratogenic properties of ethanol (e.g., [24]). Moreover, the deleterious effects of in utero ethanol exposure have been found to exist on a continuum, ranging from gross morphological defects at one extreme to more subtle cognitive/behavioral dysfunctions at the less severe end. The latter effects have established ethanol as a behavioral teratogen. Several clinical reports have demonstrated the behavioral teratogenic effects of ethanol in humans (e.g., [6, 31, 32]). These include hyperactivity, irritability, restlessness, and mental deficiencies. Likewise, animals prenatally exposed to ethanol have been shown to exhibit behavioral abnormalities similar to that reported in humans. Some of the behavioral abnormalities observed in animals prenatally exposed to ethanol include hyperactivity and increased exploratory behavior, as well as deficits in a variety of learning tasks such as discrimination and reversal learning, passive and shuttle avoidance performance, taste aversion learning, and operant conditioning situations. This growing body of literature has been reviewed by Abel [2] and, most recently, by Meyer and Riley [21]. Although there are exceptions, perhaps the most parsimonious account for many of the behavioral abnormalities that follow prenatal ethanol exposure is that these animals exhibit a response-inhibition deficit [21,26]. Poorer performance on tasks that involve withholding, or inhibiting, a response such as passive avoidance (e.g., [3, 11, 18, 26, 27])

Fetal alcohol effects

and conditioned taste aversion (e.g., [25,26]) most clearly support this hypothesis. In addition, hyperactivity and increased exploration [3, 7-9, 12, 13, 20, 33], deficits in reversal learning and spontaneous alternation [3, 17, 18, 27], increased resistance to extinction [30], and increased nippleshifting behavior [29] all lend support to the responseinhibition deficit hypothesis. Furthermore, lack of response inhibition may contribute to the enhanced performance of rats prenatally exposed to ethanol in tasks that require the subject to make an active response in order to avoid shock, i.e., free-operant and discriminated Y-maze avoidance [10, 22, 28]. With a few exceptions (e.g., extinction and nipple-shifting behavior studies), most of the above work employed aversively-motivated behavioral procedures. The purpose of the present study was to further test the response-inhibition deficit hypothesis using an appetitively-motivated task, referred to as negative contrast. This consummatory behavioral task allows for the quantification of how animals respond to an abrupt, unexpected reduction in reward. Briefly, rats shifted from a more preferred to a less preferred sucrose reward consume substantially less of the smaller reward than control animals that only experienced the less preferred reward. The suppressed performance of down-shifted animals typically returns to control levels within three to five days. Supportive data exist indicating that an active process of behavioral inhibition is involved in the depressed consummatory performance (negative contrast effect) engendered

1Requests for reprints should be addressed to Dr. Howard Becker, Research Service, VA Medical Center, 109 Bee Street, Charleston, SC 29403.

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by reward reduction [19]. In accordance with the responseinhibition deficit hypothesis, rats prenatally exposed to ethanol would be expected to be less responsive to the down-shift in reward, exhibiting a smaller negative contrast effect and/or faster recovery to control levels. METHOD Subjects Adult, male offspring obtained from Dr. Riley's laboratory at SUNY-Albany were used as subjects for behavioral testing. Prior to arriving at the VAMC-Charleston facility, the animals were exposed to their respective prenatal treatment as follows. Female Long-Evans rats (Blue Spruce Farms, Altamont, NY) were mated each evening until a seminal plug was found the following morning. On this day (day 1 of gestation), pregnant rats were weighed and individually housed in standard breeding cages in a temperature and humidity controlled nursery under a 12 hour light/dark cycle. They were then randomly assigned to one of three prenatal treatment groups. Two of these groups were provided with a liquid diet containing either 35% or 0% ethanol-derived calories (EDC) as their sole sources of nutrition on gestation days 6-20. The third group (LC) was maintained on standard lab chow and water continuously throughout gestation. Liquid diets consisted of chocolate Sustacal (Mead Johnson), vitamin and mineral diet fortification mixture (ICN Nutritional Biochernicals), ethanol (95% w/v), sucrose, and water. Sucrose was substituted isocalorically for ethanol in the 0% EDC diet and both diets provided 1.3 kcal/ml. In order to equate caloric intakes of the 35% and 0% EDC groups, a pair-feeding procedure was employed. The 35% EDC females had unrestricted access to their diets while each female in the 0% EDC group was matched to a 35% EDC female (of similar body weight) and fed the amount consumed by this female on a ml/kg body weight/day basis. Females were weighed every five days to more precisely control pair-feeding. At birth (day 0), all pups were inspected for gross physical abnormalities, weighed, and litters randomly culled to l0 (of equal sex when possible). Offspring were housed with their biological mother until day 21, at which time they were weaned and group housed according to sex and prenatal treatment condition. At approximately 40-45 days of age, male offspring were shipped to the VAMC-Charleston facility where they were individually housed in a temperature and humidity controlled colony room maintained on a 12 hour light/dark cycle (lights on at 0600). The rats were acclimated to this facility, with food and water freely available, for approximately 3-4 weeks prior to testing. Starting at approximately 60-70 days o f age, all rats were maintained at 82% of their free-feeding body weight by single daily feeding. Animals were maintained on this restricted feeding regimen, with water continuously available, throughout the experiment. Apparatus Subjects were tested in five identical metal cages (24.5x 17.5x 18 cm). A centrally located hole 1 cm in diameter and 7 cm above the floor was present on one side of each of the cages. A graduated cylinder was placed outside the chamber such that the orifice of the drinking spout was centered in the hole and flush with the outside wall of the cage. The testing cages were situated in sound-attenuating cham-

bers with dim red light and equipped to present white noise. Licking responses were monitored and recorded by programmed solid-state circuitry interfaced with electromechanical counters (Coulbourn Instruments, Lehigh Valley, PA). Procedure Prior to the start of the experiment, rats were habituated to the testing situation by being placed in the chambers for five minutes. During this habituation phase, which consisted of three daily sessions, sucrose solutions were not available. This procedure has been found to facilitate consumption of the sucrose solutions once they are introduced in the test chambers. Following the three day habituation period, rats from each of the three prenatal treatment groups were randomly divided into two groups: shifted and unshifted. With a single exception, no more than one animal from a given litter was represented in the same group. Two littermates were included in the 0% EDC shifted group. Shifted animals received access to a 32% sucrose solution for 10 days (designated as the pre-shift period) and then a 4% solution for four post-shift days. The remaining animals served as unshifted controls, receiving 4% sucrose on all days of the experiment. It is important to note that during the post-shift period, all animals received the same reward (4% sucrose). The difference between shifted and unshifted animals was that only the former group had prior experience with a more preferred reward (32% sucrose). The 14 daily sessions consisted of five minutes access to the appropriate sucrose solution, timed from the first lick. Sucrose solutions (w/v) were prepared from commercial grade cane sugar and tap water, 24 hours before each session, and presented at room temperature. The experiment was conducted in three replications. RESULTS Maternal and Pup Data The computation of maternal and pup data was based on 11, 13, and 12 litters from the 35% EDC, 0% EDC, and LC groups, respectively. Percent maternal weight gain during pregnancy was 30.8%, 31.5%, 38.5% for dams in the 35% EDC, 0% EDC, and LC groups, respectively. A N O V A on these data revealed a significant effect of Prenatal Treatment, F(2,33)=6.23, p<0.01. Subsequent analysis with Fisher's Least Significant Difference (LSD) test indicated the LC dams gained more weight than the 35% EDC and 0% EDC groups, which did not differ from each other (p<0.05). Analysis of gestation length and litter size revealed no significant differences between prenatal treatment groups. Females in the 35% EDC group consumed an average (-+SEM) of 12.64 (+_0.32) g/kg/day ethanol. Although there was a tendency for ethanol-exposed offspring to weigh less than controls at birth, this effect did not reach statistical significance. However, A N O V A revealed a significant effect of Prenatal Treatment on adult (60--80 days of age) body weight of animals just prior to behavioral testing, F(2,58)=4.85, p<0.05. Mean body weights ( _ S E M ) for the 35% EDC, 0% EDC, and LC groups were 274.3 (+_ 16.5), 302.2 (+_9.2), and 320.9 (-+10.5), respectively. Subsequent decomposition of this effect indicated the ethanol-exposed offspring weighed less than the two control groups, which did not significantly differ from each other (by LSD test, p <0.05). This difference in body weight was consistent for

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FIG. 1. Mean licks for 35% EDC, lY~ EDC, and LC offspring as a function of sucrose condition (shifted vs. unshifted) and test day. Bars represent SEM. both experimental groups (shifted and unshifted), as indicated by a non-significant interaction term ( F < 1.0).

Response to Reward Reduction (Negative Contrast) Behavioral data is based on 10, 11, and 11 shifted and unshifted animals derived from the 35% EDC, 0% EDC, and LC prenatal treatment groups, respectively. Subjects that did not make at least 500 licks by the eighth day of the experiment were not included in the analyses. Figure 1 illustrates the data obtained from behavioral testing. As can be seen, during the pre-shift period (days 1-10) all groups exhibited a typical acquisition function, with lick frequencies stabilizing in 8--10 days. During this pre-shift period, animals receiving 32% sucrose licked at a higher rate than animals receiving the 4% solution, F(1,59)=23.14, p<0.01. This greater consumption by shifted animals in comparison to unshifted controls did not vary as a function of prenatal treatment ( F s < l . 0 ) .

During the post-shift period, rats shifted from the 32% to 4% sucrose solution consumed substantially less of the latter than unshifted controls, F(1,59)=27.50, p<0.01. This negative contrast effect gradually diminished over the four postshift days, as indicated by a significant Sucrose Concentration × Day interaction, F(3,177)=24.08, p<0.01. Decomposition of this effect revealed negative contrast to be reliable on the first three post-shift days, i.e., unshifted rats licked more than shifted animals on these days (by LSD test, p<0.05). While there was a numerical tendency for ethanolexposed offspring to exhibit a smaller negative contrast effect and recover at a faster rate than controls, this effect did not reach statistical significance. Figure 2 shows the data for shifted animals expressed as a percent of the control (unshifted) response rate during the post-shift period. The gradual recovery from suppressed performance engendered by reward reduction is supported by a significant main effect of Day, F(3,87) = 37.46, p <0.01. Again, although the 35% EDC group appears to have responded at a higher rate than controls (0% EDC and LC groups), this effect was not significant.

DISCUSSION The purpose of this experiment was to determine if prenatal exposure to ethanol influences the manner in which rats respond to an appetitive behavioral situation where the rewarding value of a stimulus (sucrose solution) is abruptly and unexpectedly reduced. The data indicate that rats prenatally exposed to ethanol respond to two sucrose solution rewards in a similar fashion as controls. That is, during the pre-shift phase of the experiment, all treatment groups exhibited a similar acquisition function, with shifted animals consuming more 32% sucrose than unshifted rats that received the 4% solution. During the post-shift phase of the study, all treatment groups exhibited a significant negative contrast effect (unshifted rats consumed more 4% sucrose

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than shifted animals). While the data are suggestive that this effect (negative contrast) was smaller for e t h a n o l - e x p o s e d rats (particularly o v e r the first three post-shift days; see Fig. 2), this o u t c o m e did not a c h i e v e statistical significance. The lack of a robust (significant) effect may be due to the fact that the offspring tested in this study w e r e of adult age. Perhaps larger group differences m a y be o b s e r v e d in y o u n g e r animals since m a n y behavioral abnormalities related to prenatal ethanol e x p o s u r e h a v e been noted to be m o r e robust in y o u n g e r offspring (e.g., [3, 8, 17]), although there are exceptions (e.g., [1, 4, 23]). Finally, it should be noted that negative contrast may be considered a limiting case o f extinction b e h a v i o r (partial rather than c o m p l e t e reward reduction) (e.g., [14]). In fact, both situations h a v e b e e n found to be stressful as e v i d e n c e d by elevated plasma c o r t i c o s t e r o n e levels (e.g., [15,16]). In this light, it is interesting that prenatal e x p o s u r e to ethanol has b e e n found to h a v e varying effects on extinction behavior. F o r e x a m p l e , in an appetitively-motivated straight-alley task, with the reward being access to the mother, rats prenatally e x p o s e d to ethanol p e r f o r m e d no differently than con-

trols during extinction [5]. In contrast, prenatal e x p o s u r e to ethanol has b e e n found to increase resistance to extinction following operant conditioning [30] and classical conditioning ( C E R paradigm) [9]. W h e t h e r the different results of these studies, as well as the results from the present study, are due to varying degrees of a v e r s i v e n e s s (or s o m e o t h e r procedural factor) is not clear at present. Studies are currently being c o n d u c t e d to investigate the potential influence of prenatal ethanol e x p o s u r e on the stress response (plasma c o r t i c o s t e r o n e levels) to both partial (negative contrast) and c o m p l e t e (extinction) reward reduction.

ACKNOWLEDGEMENTS The authors would like to thank Beth Bickerstaff and Mary Ann Hannigan for their technical assistance in various aspects of this project, and Lucille von Kolnitz for her assistance in the preparation of the manuscript. This research was supported by funds from the National Institute on Alcohol Abuse and Alcoholism, grant numbers AA006893, AA00077, and AA07029, and the Veterans Administration.

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ON BEHAVIOR RESPONSE

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