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Effects of prenatal nutrition on learning and motivation in rats
Physiology & Behavior, Vol. 22, pp. 1067-1071. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
Effects of Prenatal Nutrition on Learning and Motivation in R a t s I D O U G L A S P. P E T E R S
Department of Psychology, University of North Dakota, Grand Forks ND 58202 (Received 30 O c t o b e r 1978) PETERS, D. P. Effects o f prenatal nutrition on learning and motivation in rats. PHYSIOL. BEHAV. 22(6) 1067-1071, 1979.--The results of two learning experiments revealed that adult male rats whose dams suffered either prenatal zinc deficiency or undernutrition showed, as compared to rats whose dams were fed ad lib, normal diets during pregnancy (1) more resistance to extinction following partial reinforcement, and (2) a faster occurring negative contrast effect when shifted from large to small reward. An incentive-motivation interpretation of the data suggests that the nutritionally deprived and normal animals differ with respect to the incentive value of the consummatory reward. This effect appears to be long-term. Prenatal nutrition Prenatal zinc deficiency reinforcement effects Prenatal undernutrition
Intra-uterine nutrition Incentive motivation
THE NUTRITION literature indicates that the diet of rats during prenatal and early infancy periods can be particularly important in the development of the organism. Observed biochemical (e.g. [9, 11, 43, 45]) and maturational abnormalities (e.g. [1, 2, 9, 12, 26, 41]) have been attributed to prior conditions of nutritional deficiency. In the last decade, increasing attention has been focused on the behavioral effects of malnutrition and undernutrition. A number of investigators have been concerned with the performance of nutritionally deprived rats on learning tasks. Among recent fmdings, greater errors in the Hebb-Williams maze [1, 6, 7, 47], faster running speeds and increased errors in a multiple-T maze [41], and poorer discrimination performance [5] have been observed in rats which suffered early nutritional deprivation. A common interpretation of these findings is that malnutrition and undernutrition impair general learning ability or cognitive functioning of the rat. The present investigation examined an alternative hypothesis which posits an incentive-motivation explanation for nutrition deprived vs normal rat differences seen in many learning-type experiments. Several studies have reported finding abnormal metabolic patterns [24,25] and lower efficiency of food utilization [4,20] in rats with a history of prenatal or early postnatal nutritional deficiency. This is important for its raises the possibility that the value of the consummatory rewards used in a number of learning experiments may be different for nutritionally deprived and normal-diet animals. If so, the performance differences observed when these rewards are employed should be perhaps attributed to incentive-motivation factors, rather than to deficits in intellectual ability. To assess the possible influence of undernutrition and malnutrition on the incentive value of consummatory re-
Trace elements Contrast effects
Learning
Partial
wards, two well documented phenomena in animal learning were studied: (a) the partial reinforcement extinction effect (PREE), increased resistance to extinction following intermittent vs continuous reinforcement (for review see [37]), and (b) the negative contrast effect (NCE), a reduction in performance when shifted from large to small reward that is below the response level of controls which always receive small reward (for review see [3, 10, 29]). The PREE and NCE were selected because they have been shown to be sensitive to reward-incentive manipulations, and therefore, should be useful in detecting incentive-motivation difference between nutritionally deprived and normal diet animals, assuming they do in fact exist. The specific type of malnutrition chosen for this study was prenatal zinc deficiency. Recently, nutritional researchers have become interested in the effects of dietary zinc. Rats which experience interuterine zinc deficiency show a reduction in brain size as fetal [30,39] and neonatal [40] animals. There have also been reports of biochemical aberations, e.g., impaired DNA synthesis in the brains of zinc deprived rats [22]. The limited number of behavioral studies that have been conducted indicate an increase in aggression [15, 16, 17, 33] and deficits in avoidance learning [18, 19, 28] for post-infant rats which suffered prenatal zinc deficiency. This report explores further the long-term effects of interuterine zinc deficiency on behavior, specifically foodreinforced, instrumentally conditioned behavior. EXPERIMENT 1 The first experiment tested for PREE differences among groups of adult rats which had received zinc deprived, undernourished, or normal prenatal diets. All of these animals
1This research was supported in part by the United States Department of Agriculture Cooperative Agreement 12-14-100-11, 178, Amendment 1.
Copyright © 1979 Brain Research Publications Inc.~0031-9384/79/061067-05502.00/0
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PETERS
were given partial reinforcement during acquisition training. When shifted to extinction their performance was compared to a normal-control group which had received continuous reinforcement during the acquisition period preceding extinction. METHOD
A trial was initiated by placing a rat in the start box. After a 4 sec orientation, the start door was opened. When the rat entered the goal box, a retrace door was lowered. The rat was removed immediately after consumption of the reward or 5 sec following nonreward. All latencies were converted to speed scores for analyses. Finally, every animal from each group was weighed at the start and termination of the experiment.
Animals The nutritional manipulations are similar to those described in earlier reports [11,29]. On Day 14 of pregnancy, 8 Long-Evans dams (Z) were fed a biotin-enriched 20% sprayed egg white diet which consisted of less than 1 /zg zinc/g, plus deionized water. Since anorexia results from zinc deficiency, 8 additional dams were given the quantity of diet consumed by a Z dam pair-mate. The pair-fed (P) animals were also allowed to drink water containing 50/xg zinc per ml. A third group of dams (A) had ad lib access to the preceding diet with the supplemented water. On Day 20 of pregnancy, all dams received ad lib Purina Laboratory Chow plus tap water. All litters were reduced in size to the 9 most healthy pups in the litter 24 hr following delivery. On Day 21 the pups were weaned, and then housed in plastic cages with two or three same-sex animals per cage in a temperature and humidity controlled room. At age 50 days, the four groups used in this experiment were determined by randomly selecting 8, 8 and 16 male pups from the Z, P, and A dams, respectively, with the restriction that each litter contribute at least one male. The 16 rats from the A dams were then divided into two equal groups, A and CR, with groups Z and P already formed. Following this, all experimental animals were individually housed in stainless steel cages.
RESULTS
Since each of the separate runway measures yielded similar graphical and statistical results, only the reciprocated total traversal time scores are presented. This also applies to Experiment 2. Figure 1 presents group mean total speeds as a function of blocks of eight trials.
Acquisition In looking at Fig. 1, all four groups show an increase in running speeds with acquisition training. Although it appears graphically that group A ran slower than the other groups, an analysis of variance of the total acquisition data revealed only a significant effect of trial blocks (F(6,168)=24.08, p<0.001). An additional variance analysis over the last 8 trials of the acquisition period (Block 7) failed to show a significant effect of groups (p>0.05). Thus, all groups were performing comparably at the completion of acquisition training.
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A conventional straight, black runway (120×9×9 cm) covered by a clouded Plexiglas ceiling was used. Photocell clock circuitry provided traversal times over three 30 cm segments of the runway. A fan mounted on the rear exterior wall of the goal box extracted air from the alley via 0.3 cm holes.
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Procedure The rats were 90 days old at the start of the experiment. Seven days prior to the first training day, all animals were placed on a 23 hr food deprivation schedule, which was maintained throughout testing. The animals experienced two phases of training, acquisition and extinction. During acquisition, groups Z, P, and A received 50% partial reward (i.e., half rewarded and half nonrewarded trials on a semi-random basis) for running to the goal box, while group CR received 100% continuous reward. On rewarded trials the rats were given seven 45 mg Noyes pellets. Following 56 acquisition trials all groups received a total of 40 extinction trials in which reinforcement was withheld. Trials were administered to 4 squads composed of 8 rats each, 2 from each experimental group, at a rate of 4 trials/day during both acquisition and extinction. One entire squad received its daily trials before the next squad received its trials. Within squads each animal received its first daily trial before any animal received its second trial, etc. The intertrial interval was approximately 5 min. Running order between and within squads remained fixed throughout the study.
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FIG. 1. Mean total speeds for each group as a function of blocks of eight trials. Groups Z, P, and A received partial reward during acquisition, while group CR received continuous reinforcement.
Extinction It is clear from Fig. 1 that running speeds were depressed for all rats during extinction. The animals noticably slowed
PRENATAL NUTRITION, LEARNING AND MOTIVATION down with continued extinction. Furthermore, groups Z and P appear to show the most resistance to extinction and group CR the least. An analysis of variance of the total extinction data yielded a reliable effect of groups (F(3,28)=6.48, p<0.01) and trial blocks (F(4,112)=9.96,p <0.01. The groups xblocks interaction did not reach statistical significance. Pair comparisons (t-tests) of performance levels combined over Blocks 9 and 10, where group differences appeared to be the greatest, revealed that group CR can reliably (p <0.01) slower than the other three groups, thus indicating a PREE for Z, P, and A animals. Group A ran significantly (p<0.05) slower than groups Z and P. No other comparisons were statistically reliable (p's>0.05).
Body Weight On the fh'st day of testing the mean weights of the Z, P, A and CR animals were 244, 236, 247 and 237 g, respectively. The mean weights for these groups on the last day of testing were 241,243, 244 and 233 g, respectively. An analysis of variance of this data yielded no significant effects of groups, days, or groups x days interaction (p's>0.05). EXPERIMENT 2 Evidence for long-term behavioral effects of prenatal malnutrition (group Z) and undernutrition (group P) was provided by the results of the first experiment. Groups Z and P received the same schedule of partial reinforcement as Group A, the normal-diet animals, during acquisition training, but they displayed significantly more response persistance during the extinction period. All three groups exhibited a PREE, however, the effect was greatest for the previously nutritionally deprived rats. Experiment 2 was run to determine if similar nutrition effects would be obtained with the NCE. Z, P, and A animals were trained on large reward and then shifted to small reward. To measure the occurrence of an NCE, their postshift performances were compared to a control group that always received small reward.
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were shifted from 7 to 1 pellet reward. A total of 40 trials were given during the postshift phase. A small reward control group, C, received a 1 pellet reward during both preshift and postshift training. The manner of administration of trials to squads and individual trial procedures were identical to those of Experiment 1. RESULTS Group mean total speeds are plotted for each group as a function of blocks of 8 trials in Fig. 2.
Preshift It is clear from Fig. 2 that all groups increased running speeds across trials. It also appears that group C ran the slowest during preshift, while the other 3 groups performed comparably. An analysis of variance of the total preshift data did not yield a significant effect of groups. Only an effect of blocks was statistically reliable (F(6,168)= 17.47, p<0.001). However, analyses (t-tests) of the data combined over trial blocks 3-4 revealed that group C did significantly differ (p<0.01) from groups Z, P, and A. No other group comparisons were significant (p's>0.05).
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Z, P, and A dams, identical to those of Experiment 1, contributed male offspring to this study. Groups Z and P consisted of 8 animals each selected from Z and P dams. Groups A and C were comprised of 8 rats each taken from normal-diet (A) mothers. The group assignment procedure was the same as that described for Experiment 1, as were the housing conditions.
Apparatus The apparatus was the same as that of Experiment I, i.e., conventional straight runway with food reward baited in the goal region.
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FIG. 2. Mean total speeds for each group as a function of blocks of eight trials. Groups Z, P, and A were shifted from 7 to 1 pellet reward, while group C always received a 1 pellet reward.
Procedure The animals were 90 days old at the beginning of the experiment. A 23 hr food deprivation schedule was initiated 7 days prior to, and maintained throughout testing. All groups received two phases of training, preshift and postshift. During the preshift period (56 trials), groups Z, P, and A were reinforced with seven 45 nag Noyes pellets for traversing the runway. Following this period, these animals
Postshift Following the shift from 7 to 1 pellet reward in Block 8, groups Z, P, and A showed a depression effect or NCE, i.e., they all ran more slowly to 1 pellet reward than did the control group, C, trained and maintained on the small reward. The NCE developed immediately for groups Z and P on Block 8, while the NCE displayed by group A occurred
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PET E R 5
later on Block 9. Variance analysis of the data combined over Blocks 8-9 for all groups yielded only a significant effect of groups (F(3,28)=4.68, p<0.01). Pair comparisons (ttests) of separate performance levels at Blocks 8 and 9 revealed that groups Z and P reliably, differed from groups A and C (p<0.01) at Block 8, while Z, P, and A animals all significantly differed from group C (p<0.01), but not from each other, on Block 9. These were the only statistically reliable differences. By the end of the postshift period, the 4 groups showed comparable levels of responding to the l pellet reward. Analysis of variance of the data for Blocks I l vs. 12 yielded no significant effects ( F < 1).
Body Weight An analysis of variance of body weights for groups Z, P, A and C on the first and last day of testing revealed, as was seen in Experiment 1, no significant group differences, nor were there reliable effects of days or g r o u p s × d a y s interaction (p's>0.05). GENERAL DISCUSSION Animals of this study which received either prenatal undernutrition or malnutrition showed, as compared to normal-diet controls, a greater PREE and N C E when tested as adults. These results confirm and extend earlier findings [1, 6, 7, 32, 47] which report performance differences between malnourished and normal animals on learning tasks involving consummatory rewards. The fact that there were no differences in responding for groups Z, P, and A during the first phase of instrumental conditioning in Experiment 1, during acquisition trials, and in Experiment 2, during preshift trials, is not surprising. Previous investigators [18, 34, 38] employing Z, P, and A rats with nutritional backgrounds identical to those of this study have not found, with one exception [28], any group differences in the acquisition of a food reinforced, simple instrumental response, similar in type to those conditioned in this report. Only when reinforcement conditions were altered did nutrition effects surface in the present investigation. The Z, P, and A rats of Experiment 1 experienced a change from partial reinforcement to nonreinforcement (extinction), while the animals of Experiment 2 were shifted from large to small reward. Following these reward charges, the animals from groups Z and P displayed a greater PREE in Experiment 1 and a faster occurring N C E in Experiment 2, as compared to the rats from the A groups. In light of past research on the NCE and PREE, the present performance differences seen between the previously nutritionally deprived (Z and P) and nondeprived (A) animals may reflect differences in incentive values of the consummatory rewards for these groups of animals. Z, P, and A rats received an identical 50% partial reward schedule in Experiment 1 during acquisition and they displayed nondifferential running speeds. However, during extinction the Z and P rats exhibited the most response persistance. It is documented that resistance to extinction increases as the rewardincentive values in partial reward (PR) schedules increase [13, 27, 36, 42]. F o r example, animals trained on a 50% PR schedule of 15 or 0 pellets (15/0) will show more resistance to extinction than animals which receive 7/0 PR during acquisition. The nominal acquisition reward for groups Z and P of
Experiment 1 was 7/0 PR, but as their extinction performance suggests, a higher reward-incentive may have been associated with this schedule, perhaps something comparable to the incentive value of a 15/0 PR schedule. This could explain why these two groups showed more resistance to extinction than group A. An incentive-motivation interpretation is also amenable to the findings of Experiment 2, where groups Z and P displayed an immediate NCE with the absolute magnitude being greatest for Z rats. A number of prior NCE studies [23, 3 I, 35] have indicated that the development and magnitude of the NCE increases as the preshift reward values increase. Groups Z, P, and A all received 7 pellets reward on preshift trials, but only the Z and P rats displayed a rapid NCE when shifted to the smaller 1 pellet reward. This finding suggests that the incentive value attributed to the 7 pellet preshift reward was greater for the Z and P animals as compared to the normal, A, rats, and consequently, groups Z and P experienced a larger incentive shift during the postshift phase. If prenatal conditions of undernutrition and zinc deficiency alter the incentive-motivation of consummatory rewards for adult rats, the effect, as the present data implies, is one of heightening the incentive values of food rewards experienced over trials. A recent report [14] which used Z, P and A rats identical to the present animals has produced evidence consistent with this observation, i.e., the Z and P rats were significantly more motivated for food reinforcement than normal-diet animals in a simple instrumental conditioning task. This heightening of reward-incentives for nutritionally deprived animals has also been found with primates. Zimmerman [46], for example, in studying the effects of protein malnutrition on complex learning in rhesus monkeys, has obtained behavioral differences between malnourished and normal animals, with the deprived monkeys being superior in some cases. These differences were attributed to the greater incentive value placed on food reinforcement by the malnourished animals. The same interpretation was applied to the results of a later study [44] where protein-deficient monkeys showed dominance over normal-diet monkeys in food-competition situations. The malnourished animals were clearly more motivated for food. In contrast to the above, one report [8] in the literature has found what appears to be lower food incentives for offspring of underfed mothers in comparison to rats from normal-diet mothers. However, since this study employed electric shock concurrently with food reinforcement, it is difficult to separate performance differences based on motivational factors associated with aversive stimulation vs. appetitive rewards. The use of shock in this situation could be critical, since recent experiments [15, 16, 17] with Z, P, and A rats have discovered behavioral (e.g., aggression) differences among these groups when shock is administered. Accumulating data, like that of the present investigation, strongly suggest that in experiments where nutritionally deprived animals show patterns of behavior that differ from normal controls, these differences should be assigned to motivational factors, rather than to deficits in cognitive functioning or learning ability. Nutritional studies employing appetitive rewards, e.g., food reinforcement, can be particularly susceptable to incentive differences among nutrition groups, as the results of this study would indicate.
PRENATAL
NUTRITION,
LEARNING
AND MOTIVATION
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REFERENCES 1. Baird, A., E. M. Widdowson and J. J. Cowley. Effects of calorie and protein deficiencies early in life on the subsequent learning ability of rats. Br. J. Nutr. 25: 391-403, 1971. 2. Barnes, R., A. Moore, I. Reid and W. Pond. Effects of food deprivation on behavior patterns. In: Malnutrition, Learning and Behavior, edited by N. Scrimshaw and J. Gorden. Cambridge: M.I.T. Press, 1%8, pp. 203-217. 3. Black, R. W. Shifts in magnitude of reward and contrast effects in instrumental and selective learning: A reinterpretation. Psychol. Rev. 75: 114-126, 1968. 4. Blackwell, B. N., R. Q. Blackwell, T. T. Yu, Y. S. Weng and B. F. Chow. Further studies on growth and feed utilization in progeny of underfed mother rats. J. Nutr. 97: 79-84, 1%9. 5. Bresler, D. E., G. Ellison and S. Zamenhof. Learning deficits in rats with malnourished grandmothers. Devl Psychobiol. 8: 315-323, 1975. 6. Cowley, J. J. and R. D. Griesel. The development of a second generation of low protein rats. J. genet. Psychol. 103: 233-242, 1%3. 7. Cowley, J. J. and R. D. Griesel. The effect on growth and behavior of rehabilitating first and second generation low protein rats. Anita. Behav. 14: 506-517, 1%6. 8. Cravens, R. W. Effect of maternal undernutrition on offspring behavior: Incentive value of a food reward and ability to escape from water. Devl Psychobiol. 7: 61-69, 1974. 9. Culley, W. J. and R. O. Lineberger. Effect of undernutrition on the size and composition of the rat brain. J. Nutr. 96: 375-381, 1%8. 10. Dunham, P. J. Contrasted conditions of reinforcement: A selective critique. Psychol. Bull. 69: 295-315, 1%8. 11. Fosmire, G., Y. Y. Al-Ubaidi and H. H. Sandstead. Some effects of postnatal zinc deficiency on developing rat brain. Pediat. Res. 9: 89-93, 1975. 12. Fra6kov~, S. and R. H. Barnes. Effect of malnutrition in early life on avoidance conditioning and behavior of adult rats. J. Nutr. %: 485-493, 1%8. 13. Gonzalez, R. C. and M. E. Bitterman. Spaced-trials partial reinforcement effect as a function of contrast. J. comp. physiol. Psychol. 67: 94-103, 1969. 14. Halas, E. S., P. A. Burger and H. H. Sandstead. Food motivation of rehabilitated malnourished rats. Paper presented at Midwestern Psychol. Assoc. meeting, Chicago, May, 1978. 15. Halas, E. S., M. J. Hanlon and H. H. Sandstead. Intrauterine nutrition and aggression. Nature 257: 221-222, 1975. 16. Halas, E. S., G. M. Reynolds, M. Rowe, M. Heinrich and M. Pirc. Comparison of frequency, intensity and duration of aggressive responses in rats. Physiol. Behav. 18: 975-977, 1977. 17. Halas, E. S., G. M. Reynolds and H. H. Sandstead. Intrauterine nutrition and its effects on aggression. Physiol. Behav. 19: 653-661, 1977. 18. Halas, E. S., M. C. Rowe, O. R. Johnson, J. M. McKenzie and H. H. Sandstead. Effects of intrauterine zinc deficiency on subsequent behavior. In: Trace Elements in Human Health and Disease: Volume 1, Copper and Zinc, edited by A. S. Prasad. New York/London: Academic Press, 1976, pp. 327-343. 19. H~as, E. S. and H. H. Sandstead. Some effects of prenatal zinc deficiency on behavior of the adult rat. Pediat. Res. 9" 94-97, 1975. 20. Hsueth, A. M., R. Q. Blackwell and B. F. Chow. Effect of maternal diet in rats on feed consumption of the offspring. J. Nutr. 100: 1157-1164, 1970. 21. Hulse, S. H, Amount and percentage of reinforcement and duration of goal confinement in conditioning and extinction. J. exp. Psychol. 56: 48-57, 1958. 22. Hurley, L. S. Zinc deficiency in the developing rat. J. Clin. Nutr. 22: 1332-1339, 1%9. 23. Ison, J. R., D. H. Glass and H. B. Daly. Reward magnitude changes following differential conditioning and partial reinforcement. J. exp. Psychol. 81: 81-88, 1%9.
24. Lee, C. J. and B. F. Chow. Protein metabolism in the offspring of underfed mother rats. J. Nutr. 87: 439--443, I%5. 25. Lee, C. J. and B. F. Chow. Metabolism of proteins by progency of underfed mother rats. J. Nutr. 94: 20-26, 1968. 26. Levitsky, D. A. and R. H. Barnes. Nutritional and environmental interaction in the behavioral development of the rat: Long-term effects. Science 176: 68-71, 1972. 27. Likely, D., L. Little and N. J. Mackintosh. Extinction as a function of magnitude and percentage of food or sucrose reward. Can. J. Psychol. 25: 130-137, 1971. 28. Lokken, P. M., E. S. Halas and H. H. Sandstead. Influence of zinc deficiency on behavior. Proc. Soc. exp. Biol. Med. 144: 680-682, 1973. 29. McHose, J. H. Relative reinforcement effects: $1/$2 and SflSI paradigms in instrumental conditioning. Psychol. Rev. 77: 135146, 1970. 30. McKenzie, J. M., G. J. Fosmire and H. H. Sandstead. Zinc deficiency during the later third of pregnancy: Effects on fetal rat brain, liver and placenta. J. Nutr. 105: 1466-1475, 1975. 31. Mikulka, P. J., R. Lehr and W. B. Pavlik. Effect of reinforcement schedules on reward shifts. J. exp. Psychol. 74: 57-61, 1%7. 32. Ottinger, D. R. and G. Tanabe. Maternal food restriction: Effects on offspring behavior and development. Devl Psychobiol. 2: 7-9, 1%8. 33. Peters, D. P. Effects of prenatal nutritional deficiency on affiliation and aggression in rats. Physiol. Behav. 20: 35%362, 1978. 34. Peters, D. P. Effects of prenatal nutritional deficiency on discrimination learning in rats: Acquisition and retention. Psychol. Rep., in press. 35. Peters, D. P. and J. H. McHose. Effects of varied preshift reward magnitude on successive negative contrast effects in rats. J. comp. physiol. Psychol. 86: 85-95, 1974. 36. Ratliff, R. G. and A. R. Ratliff. Runway acquisition and extinction as a joint function of magnitude of reward and percentage of reward acquisition trials. Learn. Motivat. 2: 28%295, 1971. 37. Robbins, D. Partial reinforcement: A selective review of the alleyway literature since 1961. Psychol. Bull. 76: 415-431, 1971. 38. Rowe, M. C. Physiological and behavioral effects of zinc deficiency in rats. M.A. Thesis, University of North Dakota, 1974. 39. Sandstead, H. H., G. J. Fosmire, J. M. McKenzie and E. S. Halas. Zinc deficiency and brain development in the rat. Fedn Proc. 34: 86-88, 1975. 40. Sandstead, H. H., D. D. Gillespie and R. N. Brady. Zinc deficiency: Effect on brain of the suckling rat. Pediat. Res. 6: 11% 125, 1972. 41. Simonson, M. and B. F. Chow. Maze studies on progeny of underfed mother rats. J. Nutr. 100: 685-690, 1970. 42. Wagner, A. R. Effect of amount and percentage of reinforcement and number of acquisition trials on conditioning and extinction. J. exp. Psychol. 62: 234-242, 1961. 43. Winick, M. and A. Noble. Cellular response in rats during malnutrition at various ages. J. Nutr. 89: 300-307, 1%6. 44. Wise, L. A. and R. R. Zimmerman. The effects of protein deprivation on dominance as measured by shock avoidance competition and food competition. Behav. Biol. 9: 317-329, 1973. 45. Zamenhof, S. E., E. van Marthens and F. Margolis. DNA (cell number) and protein in neonatal brain. Alteration by maternal dietary protein restriction. Science 160: 322-323, i%8. 46. Zimmerman, R. R. Effect of age, experience and malnourishmerit on object retention in learning set. Percept. Mot. Skills 28: 867-876, 1%9. 47. Zimmerman, R. R. and A. M. Wells. Performance of malnourished rats on the Hebb-Williams closed-field maze learning task. Percept. Mot. Skills 33: 1043-1050, 1971.
Effects of Prenatal Nutrition on Learning and Motivation in R a t s I D O U G L A S P. P E T E R S
Department of Psychology, University of North Dakota, Grand Forks ND 58202 (Received 30 O c t o b e r 1978) PETERS, D. P. Effects o f prenatal nutrition on learning and motivation in rats. PHYSIOL. BEHAV. 22(6) 1067-1071, 1979.--The results of two learning experiments revealed that adult male rats whose dams suffered either prenatal zinc deficiency or undernutrition showed, as compared to rats whose dams were fed ad lib, normal diets during pregnancy (1) more resistance to extinction following partial reinforcement, and (2) a faster occurring negative contrast effect when shifted from large to small reward. An incentive-motivation interpretation of the data suggests that the nutritionally deprived and normal animals differ with respect to the incentive value of the consummatory reward. This effect appears to be long-term. Prenatal nutrition Prenatal zinc deficiency reinforcement effects Prenatal undernutrition
Intra-uterine nutrition Incentive motivation
THE NUTRITION literature indicates that the diet of rats during prenatal and early infancy periods can be particularly important in the development of the organism. Observed biochemical (e.g. [9, 11, 43, 45]) and maturational abnormalities (e.g. [1, 2, 9, 12, 26, 41]) have been attributed to prior conditions of nutritional deficiency. In the last decade, increasing attention has been focused on the behavioral effects of malnutrition and undernutrition. A number of investigators have been concerned with the performance of nutritionally deprived rats on learning tasks. Among recent fmdings, greater errors in the Hebb-Williams maze [1, 6, 7, 47], faster running speeds and increased errors in a multiple-T maze [41], and poorer discrimination performance [5] have been observed in rats which suffered early nutritional deprivation. A common interpretation of these findings is that malnutrition and undernutrition impair general learning ability or cognitive functioning of the rat. The present investigation examined an alternative hypothesis which posits an incentive-motivation explanation for nutrition deprived vs normal rat differences seen in many learning-type experiments. Several studies have reported finding abnormal metabolic patterns [24,25] and lower efficiency of food utilization [4,20] in rats with a history of prenatal or early postnatal nutritional deficiency. This is important for its raises the possibility that the value of the consummatory rewards used in a number of learning experiments may be different for nutritionally deprived and normal-diet animals. If so, the performance differences observed when these rewards are employed should be perhaps attributed to incentive-motivation factors, rather than to deficits in intellectual ability. To assess the possible influence of undernutrition and malnutrition on the incentive value of consummatory re-
Trace elements Contrast effects
Learning
Partial
wards, two well documented phenomena in animal learning were studied: (a) the partial reinforcement extinction effect (PREE), increased resistance to extinction following intermittent vs continuous reinforcement (for review see [37]), and (b) the negative contrast effect (NCE), a reduction in performance when shifted from large to small reward that is below the response level of controls which always receive small reward (for review see [3, 10, 29]). The PREE and NCE were selected because they have been shown to be sensitive to reward-incentive manipulations, and therefore, should be useful in detecting incentive-motivation difference between nutritionally deprived and normal diet animals, assuming they do in fact exist. The specific type of malnutrition chosen for this study was prenatal zinc deficiency. Recently, nutritional researchers have become interested in the effects of dietary zinc. Rats which experience interuterine zinc deficiency show a reduction in brain size as fetal [30,39] and neonatal [40] animals. There have also been reports of biochemical aberations, e.g., impaired DNA synthesis in the brains of zinc deprived rats [22]. The limited number of behavioral studies that have been conducted indicate an increase in aggression [15, 16, 17, 33] and deficits in avoidance learning [18, 19, 28] for post-infant rats which suffered prenatal zinc deficiency. This report explores further the long-term effects of interuterine zinc deficiency on behavior, specifically foodreinforced, instrumentally conditioned behavior. EXPERIMENT 1 The first experiment tested for PREE differences among groups of adult rats which had received zinc deprived, undernourished, or normal prenatal diets. All of these animals
1This research was supported in part by the United States Department of Agriculture Cooperative Agreement 12-14-100-11, 178, Amendment 1.
Copyright © 1979 Brain Research Publications Inc.~0031-9384/79/061067-05502.00/0
1068
PETERS
were given partial reinforcement during acquisition training. When shifted to extinction their performance was compared to a normal-control group which had received continuous reinforcement during the acquisition period preceding extinction. METHOD
A trial was initiated by placing a rat in the start box. After a 4 sec orientation, the start door was opened. When the rat entered the goal box, a retrace door was lowered. The rat was removed immediately after consumption of the reward or 5 sec following nonreward. All latencies were converted to speed scores for analyses. Finally, every animal from each group was weighed at the start and termination of the experiment.
Animals The nutritional manipulations are similar to those described in earlier reports [11,29]. On Day 14 of pregnancy, 8 Long-Evans dams (Z) were fed a biotin-enriched 20% sprayed egg white diet which consisted of less than 1 /zg zinc/g, plus deionized water. Since anorexia results from zinc deficiency, 8 additional dams were given the quantity of diet consumed by a Z dam pair-mate. The pair-fed (P) animals were also allowed to drink water containing 50/xg zinc per ml. A third group of dams (A) had ad lib access to the preceding diet with the supplemented water. On Day 20 of pregnancy, all dams received ad lib Purina Laboratory Chow plus tap water. All litters were reduced in size to the 9 most healthy pups in the litter 24 hr following delivery. On Day 21 the pups were weaned, and then housed in plastic cages with two or three same-sex animals per cage in a temperature and humidity controlled room. At age 50 days, the four groups used in this experiment were determined by randomly selecting 8, 8 and 16 male pups from the Z, P, and A dams, respectively, with the restriction that each litter contribute at least one male. The 16 rats from the A dams were then divided into two equal groups, A and CR, with groups Z and P already formed. Following this, all experimental animals were individually housed in stainless steel cages.
RESULTS
Since each of the separate runway measures yielded similar graphical and statistical results, only the reciprocated total traversal time scores are presented. This also applies to Experiment 2. Figure 1 presents group mean total speeds as a function of blocks of eight trials.
Acquisition In looking at Fig. 1, all four groups show an increase in running speeds with acquisition training. Although it appears graphically that group A ran slower than the other groups, an analysis of variance of the total acquisition data revealed only a significant effect of trial blocks (F(6,168)=24.08, p<0.001). An additional variance analysis over the last 8 trials of the acquisition period (Block 7) failed to show a significant effect of groups (p>0.05). Thus, all groups were performing comparably at the completion of acquisition training.
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A conventional straight, black runway (120×9×9 cm) covered by a clouded Plexiglas ceiling was used. Photocell clock circuitry provided traversal times over three 30 cm segments of the runway. A fan mounted on the rear exterior wall of the goal box extracted air from the alley via 0.3 cm holes.
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Procedure The rats were 90 days old at the start of the experiment. Seven days prior to the first training day, all animals were placed on a 23 hr food deprivation schedule, which was maintained throughout testing. The animals experienced two phases of training, acquisition and extinction. During acquisition, groups Z, P, and A received 50% partial reward (i.e., half rewarded and half nonrewarded trials on a semi-random basis) for running to the goal box, while group CR received 100% continuous reward. On rewarded trials the rats were given seven 45 mg Noyes pellets. Following 56 acquisition trials all groups received a total of 40 extinction trials in which reinforcement was withheld. Trials were administered to 4 squads composed of 8 rats each, 2 from each experimental group, at a rate of 4 trials/day during both acquisition and extinction. One entire squad received its daily trials before the next squad received its trials. Within squads each animal received its first daily trial before any animal received its second trial, etc. The intertrial interval was approximately 5 min. Running order between and within squads remained fixed throughout the study.
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FIG. 1. Mean total speeds for each group as a function of blocks of eight trials. Groups Z, P, and A received partial reward during acquisition, while group CR received continuous reinforcement.
Extinction It is clear from Fig. 1 that running speeds were depressed for all rats during extinction. The animals noticably slowed
PRENATAL NUTRITION, LEARNING AND MOTIVATION down with continued extinction. Furthermore, groups Z and P appear to show the most resistance to extinction and group CR the least. An analysis of variance of the total extinction data yielded a reliable effect of groups (F(3,28)=6.48, p<0.01) and trial blocks (F(4,112)=9.96,p <0.01. The groups xblocks interaction did not reach statistical significance. Pair comparisons (t-tests) of performance levels combined over Blocks 9 and 10, where group differences appeared to be the greatest, revealed that group CR can reliably (p <0.01) slower than the other three groups, thus indicating a PREE for Z, P, and A animals. Group A ran significantly (p<0.05) slower than groups Z and P. No other comparisons were statistically reliable (p's>0.05).
Body Weight On the fh'st day of testing the mean weights of the Z, P, A and CR animals were 244, 236, 247 and 237 g, respectively. The mean weights for these groups on the last day of testing were 241,243, 244 and 233 g, respectively. An analysis of variance of this data yielded no significant effects of groups, days, or groups x days interaction (p's>0.05). EXPERIMENT 2 Evidence for long-term behavioral effects of prenatal malnutrition (group Z) and undernutrition (group P) was provided by the results of the first experiment. Groups Z and P received the same schedule of partial reinforcement as Group A, the normal-diet animals, during acquisition training, but they displayed significantly more response persistance during the extinction period. All three groups exhibited a PREE, however, the effect was greatest for the previously nutritionally deprived rats. Experiment 2 was run to determine if similar nutrition effects would be obtained with the NCE. Z, P, and A animals were trained on large reward and then shifted to small reward. To measure the occurrence of an NCE, their postshift performances were compared to a control group that always received small reward.
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were shifted from 7 to 1 pellet reward. A total of 40 trials were given during the postshift phase. A small reward control group, C, received a 1 pellet reward during both preshift and postshift training. The manner of administration of trials to squads and individual trial procedures were identical to those of Experiment 1. RESULTS Group mean total speeds are plotted for each group as a function of blocks of 8 trials in Fig. 2.
Preshift It is clear from Fig. 2 that all groups increased running speeds across trials. It also appears that group C ran the slowest during preshift, while the other 3 groups performed comparably. An analysis of variance of the total preshift data did not yield a significant effect of groups. Only an effect of blocks was statistically reliable (F(6,168)= 17.47, p<0.001). However, analyses (t-tests) of the data combined over trial blocks 3-4 revealed that group C did significantly differ (p<0.01) from groups Z, P, and A. No other group comparisons were significant (p's>0.05).
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Z, P, and A dams, identical to those of Experiment 1, contributed male offspring to this study. Groups Z and P consisted of 8 animals each selected from Z and P dams. Groups A and C were comprised of 8 rats each taken from normal-diet (A) mothers. The group assignment procedure was the same as that described for Experiment 1, as were the housing conditions.
Apparatus The apparatus was the same as that of Experiment I, i.e., conventional straight runway with food reward baited in the goal region.
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FIG. 2. Mean total speeds for each group as a function of blocks of eight trials. Groups Z, P, and A were shifted from 7 to 1 pellet reward, while group C always received a 1 pellet reward.
Procedure The animals were 90 days old at the beginning of the experiment. A 23 hr food deprivation schedule was initiated 7 days prior to, and maintained throughout testing. All groups received two phases of training, preshift and postshift. During the preshift period (56 trials), groups Z, P, and A were reinforced with seven 45 nag Noyes pellets for traversing the runway. Following this period, these animals
Postshift Following the shift from 7 to 1 pellet reward in Block 8, groups Z, P, and A showed a depression effect or NCE, i.e., they all ran more slowly to 1 pellet reward than did the control group, C, trained and maintained on the small reward. The NCE developed immediately for groups Z and P on Block 8, while the NCE displayed by group A occurred
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PET E R 5
later on Block 9. Variance analysis of the data combined over Blocks 8-9 for all groups yielded only a significant effect of groups (F(3,28)=4.68, p<0.01). Pair comparisons (ttests) of separate performance levels at Blocks 8 and 9 revealed that groups Z and P reliably, differed from groups A and C (p<0.01) at Block 8, while Z, P, and A animals all significantly differed from group C (p<0.01), but not from each other, on Block 9. These were the only statistically reliable differences. By the end of the postshift period, the 4 groups showed comparable levels of responding to the l pellet reward. Analysis of variance of the data for Blocks I l vs. 12 yielded no significant effects ( F < 1).
Body Weight An analysis of variance of body weights for groups Z, P, A and C on the first and last day of testing revealed, as was seen in Experiment 1, no significant group differences, nor were there reliable effects of days or g r o u p s × d a y s interaction (p's>0.05). GENERAL DISCUSSION Animals of this study which received either prenatal undernutrition or malnutrition showed, as compared to normal-diet controls, a greater PREE and N C E when tested as adults. These results confirm and extend earlier findings [1, 6, 7, 32, 47] which report performance differences between malnourished and normal animals on learning tasks involving consummatory rewards. The fact that there were no differences in responding for groups Z, P, and A during the first phase of instrumental conditioning in Experiment 1, during acquisition trials, and in Experiment 2, during preshift trials, is not surprising. Previous investigators [18, 34, 38] employing Z, P, and A rats with nutritional backgrounds identical to those of this study have not found, with one exception [28], any group differences in the acquisition of a food reinforced, simple instrumental response, similar in type to those conditioned in this report. Only when reinforcement conditions were altered did nutrition effects surface in the present investigation. The Z, P, and A rats of Experiment 1 experienced a change from partial reinforcement to nonreinforcement (extinction), while the animals of Experiment 2 were shifted from large to small reward. Following these reward charges, the animals from groups Z and P displayed a greater PREE in Experiment 1 and a faster occurring N C E in Experiment 2, as compared to the rats from the A groups. In light of past research on the NCE and PREE, the present performance differences seen between the previously nutritionally deprived (Z and P) and nondeprived (A) animals may reflect differences in incentive values of the consummatory rewards for these groups of animals. Z, P, and A rats received an identical 50% partial reward schedule in Experiment 1 during acquisition and they displayed nondifferential running speeds. However, during extinction the Z and P rats exhibited the most response persistance. It is documented that resistance to extinction increases as the rewardincentive values in partial reward (PR) schedules increase [13, 27, 36, 42]. F o r example, animals trained on a 50% PR schedule of 15 or 0 pellets (15/0) will show more resistance to extinction than animals which receive 7/0 PR during acquisition. The nominal acquisition reward for groups Z and P of
Experiment 1 was 7/0 PR, but as their extinction performance suggests, a higher reward-incentive may have been associated with this schedule, perhaps something comparable to the incentive value of a 15/0 PR schedule. This could explain why these two groups showed more resistance to extinction than group A. An incentive-motivation interpretation is also amenable to the findings of Experiment 2, where groups Z and P displayed an immediate NCE with the absolute magnitude being greatest for Z rats. A number of prior NCE studies [23, 3 I, 35] have indicated that the development and magnitude of the NCE increases as the preshift reward values increase. Groups Z, P, and A all received 7 pellets reward on preshift trials, but only the Z and P rats displayed a rapid NCE when shifted to the smaller 1 pellet reward. This finding suggests that the incentive value attributed to the 7 pellet preshift reward was greater for the Z and P animals as compared to the normal, A, rats, and consequently, groups Z and P experienced a larger incentive shift during the postshift phase. If prenatal conditions of undernutrition and zinc deficiency alter the incentive-motivation of consummatory rewards for adult rats, the effect, as the present data implies, is one of heightening the incentive values of food rewards experienced over trials. A recent report [14] which used Z, P and A rats identical to the present animals has produced evidence consistent with this observation, i.e., the Z and P rats were significantly more motivated for food reinforcement than normal-diet animals in a simple instrumental conditioning task. This heightening of reward-incentives for nutritionally deprived animals has also been found with primates. Zimmerman [46], for example, in studying the effects of protein malnutrition on complex learning in rhesus monkeys, has obtained behavioral differences between malnourished and normal animals, with the deprived monkeys being superior in some cases. These differences were attributed to the greater incentive value placed on food reinforcement by the malnourished animals. The same interpretation was applied to the results of a later study [44] where protein-deficient monkeys showed dominance over normal-diet monkeys in food-competition situations. The malnourished animals were clearly more motivated for food. In contrast to the above, one report [8] in the literature has found what appears to be lower food incentives for offspring of underfed mothers in comparison to rats from normal-diet mothers. However, since this study employed electric shock concurrently with food reinforcement, it is difficult to separate performance differences based on motivational factors associated with aversive stimulation vs. appetitive rewards. The use of shock in this situation could be critical, since recent experiments [15, 16, 17] with Z, P, and A rats have discovered behavioral (e.g., aggression) differences among these groups when shock is administered. Accumulating data, like that of the present investigation, strongly suggest that in experiments where nutritionally deprived animals show patterns of behavior that differ from normal controls, these differences should be assigned to motivational factors, rather than to deficits in cognitive functioning or learning ability. Nutritional studies employing appetitive rewards, e.g., food reinforcement, can be particularly susceptable to incentive differences among nutrition groups, as the results of this study would indicate.
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LEARNING
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