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Handling rat pups after early weaning
BEHAVIORAL BIOLOGY, 13, 505-509 (1975), Abstract No. 4161
BRIEF REPORT Handling Rat Pups After Early Weaning z DAVID I. WILLIAMS, GILLIAN B. BAILEY, and MICHAEL H. S. LEE
Department of Psychology, The University, Hull, England
To determine if handling infant rats acts directly to change development or whether the effect is an indirect one through the mother, pups were weaned at 14 days and the dam removed; half the fitters were handled on the next 8 days. Handling produced long-term changes in weight and behavior. Handling rat pups early in life produces gross physiological and behavioral changes both in development and in the adult animal (cf. Denenberg, 1967; Levine, 1969). The question arises as to whether this phenomenon is mediated by direct action of the stimulation on the neonate or through some indirect path (Russell, 1971). This latter situation would arise if the mother reacted to the treatment of her pups, and her subsequent behavior produced the long-term changes observed in the young (Meier and Schutzman, 1968). That there is a direct effect is not in doubt (Denenberg, 1969). But it is difficult to establish whether this momentary change results in the effects seen in the adult animal, for after stimulation the pup has to be returned to its mother and so is subject to her influence. The ideal solution is simple: remove the mother. In practice this is difficult as the mother is responsible for feeding and general care of her offspring. Artificial feeding methods (Thoman and Arnold, 1968) and early weaning techniques (Whittier and Littman, 1965) have been developed, but as these involve handling of the young they preclude a test of the infantile stimulation effect. In this study it has proved possible to use the early weaning procedure of Whittier and Littman without touching the pups in any way. So the mother was removed from the litter at 14 days after birth, 7 days before the normal weaning time. This enabled pups to be handled during the "sensitive" preweaning period without the mother, and so eliminate indirect maternal stimulation effects. 1This research was supported by grants from the Medical Research Council of Great Britain. 505 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
506
WILLIAMS, BAILEY AND LEE
Three days before the expected date of parturition 23 pregnant black hooded rats (strain PVG/C) were housed individually in opaque plastic boxes measuring 21 × 40.5 X 15 cm with wire mesh lids. The experimental room was illuminated for 16 hr per day and maintained at a temperature of 26 + I°C. At birth all litters were culled randomly to six pups and left undisturbed, except for replenishment of food and water, until Day 14 of life. On Day 14 mothers were removed and pups provided with a diet of brown wholemeal bread soaked in warm sterilized milk; water was always available and solid food (Oxoid diet 41B) was introduced on Day 19. Solid food only was provided from Day 21. Litters were allocated to either a handled (H) or nonhandled (NH) condition. The handling procedure involved removing the mother from the cage and taking each pup individually from the nest by hand and placing it in a separate compartment which measured 10 cm a in a wooden holding box; when all the pups had been removed they were replaced in the same manner; the operation took approximately 30 sec for each pup. Handling took place on days 14-21 inclusive; apart from this treatment no litter was disturbed until weighing at 25 days. At 53 + 2 days 60 rats with equal numbers of each sex from each group were tested in an open-field apparatus for three daily 3-min trims using the procedure of Williams and Russell (1972). Rats were selected evenly across litters but, because of random culling at birth giving different sex ratios within litters, numbers from each litter were not equal. The maximum number of animals from any one litter was two in each subgroup of 15; variation due to litter factors was thus small. Each rat was weighed before testing in the open field. Of the animals tested in the open-field 48 were assayed for adrenal ascorbic acid at 63 days of age using the procedure of Levine and Lewis (1959); half were subjected to a stress of three scrambled electric shocks (1.7 mA) of 30-sec duration delivered randomly in a 3-rain period prior to testing. Forty-two animals that had not been run in the open-field were tested on a standard cage-emergence procedure (Williams and Wells, 1970) at 76 days of age. Four complete litters (1H and 3NH) were destroyed prior to 21 aays as they appeared unlikely to survive. Results are presented from the remaining 19 litters (10H : 9NH); from these 4H and 6NH pups died prior to weaning. Total losses were, therefore, 10H and 24NH animals. At 25 days H animals were heavier than NH but the difference was not significant. By 50 days, however, males were significantly heavier than females (F(1,56) = 101.32, P < 0.001), and H animals significantly heavier than NH (F(1,56) = 16.14, P < 0.001). Ambulation scores in the open field showed a significant Trials X Sex × Treatment interaction (F (2,112)= 4.04, P < 0.05) which arose because H females did not show the drop-off in ambulation over time as did the other groups. The main interest here in the open-field measures lies in the index of entries into the center circle (Russell and
507
HANDLING AFTER EARLY WEANING TABLE 1 Open-Field Measuresa Entries into center
Defecation
Weight at 25 days
Weight at 50 days
Handled Male Female
35.5 3 41.87
2.31 1.38
38.96 38.07
157.87 125.20
Nonhandled Male Female
30.73 40.38
2.20 1.11
37.57 36.50
143.67 114.87
Ambulation
aEach figure is the mean over all trials. Weights are means expressed in grams.
Williams, 1973), where females made significantly more entries than males = 4.63, P < 0.05), and more importantly, H rats made significantly more entries than NH (F(1,56) = 17.71, P > 0 . 0 0 1 ) . Defecation scores showed the expected trend of males greater than females and NH greater than H but differences proved nonsignificant. Data on weight and open-field scores are shown in Table 1. On the emergence from cage measure (Rearing) a Mann-Whitney U test was used to analyze data separately for sex; H females (median 8.5 sec) reared faster than NH females (median 32 sec) (U = 37, nl = 10, n2 = 14, P < 0 . 0 5 ; 1-tailed), the male difference was in the same direction (H median 13.4 sec; NH median 30 sec) but this was not significant. For the adrenal ascorbic acid assay the mean for stressed animals was lower than for the control. While there were no significant differences between the groups in the control condition, there was in the stressed condition a significant Sex × Treatment interaction (F(1,20) = 6.81, P < 0.025). This arose because of a greater depletion of adrenal ascorbic acid in NH as compared with H females; this difference was not shown in males. The early weaning technique was successful in that the majority of pups survived. More NH than H animals were lost. This is contrary to findings in our own laboratory and elsewhere (Thoman and Levine, 1970) where, when handling is carried out with the mother present, H animals are more often lost. So differences due to handling cannot be explained on a "survival of the fittest" basis that handling selects stronger nonemotional animals by stressing and subsequently killing the weaker pups; although this hypothesis may well be relevant to some reported studies. Over-all then, as evidenced primarily by the weight measure and entries into the center of the open field, the handling treatment was effective in producing long-term changes. It is noteworthy that this procedure seemed to
(F(1,56)
508
WILLIAMS,BAILEY AND LEE
have more effect on females as shown by ambulation, rearing, and adrenal ascorbic acid depletion. These differences were found despite the fact that both groups would have been subject to stress in removing the mother at such an early date (Hofer, 1970). Also both groups would be equally liable to cool which presents problems for the hypothesis that infantile stimulation effects are mediated by hypothermia (Schaeffer, 1963). In addition, it is clear that this effect is more than habituation to handling influencing later behavioral tests (Henderson, 1966) for clear-cut physical differences in weight were found. A further problem is that an effect is obtained here with handling only in the third week postpartum. Other workers (Denenberg and Karas, 1960; Schaeffer, 1963) using the normal procedure of having the mother present failed to show an effect of handling during this period. It is tempting to speculate that in these studies the mother may have acted as a buffer to handling. But, on the other hand, there have been reports (Weininger, 1953; 1956) of handling being effective in the postweaning period, although these results have been generally discounted (Denenberg, 1964). Early weaning produces many behavioral and physiological differences which persist into adulthood (e.g., Nov~kova, 1966; 1970). The effectiveness of handling so late in the preweaning period could have resulted from an interaction with the effects of early weaning. Nevertheless the fact remains that a handling effect can be produced in the absence of the mother showing that there must be some direct effect of stimulation producing subsequent changes in development and adult behavior. This is not to say that when the mother is present her behavior is not relevant to the production of long-term changes resulting from handling; this indirect route could also be operative.
REFERENCES Denenberg, V. H. (1964). Critical periods, stimulus input, and emotional reactivity: A theory of infantile stimulation. Psychol. Rev. 71, 335-351. Denenberg, V. H. (1967). In D. C. Glass (Ed.), "Biology of Behavior: Neurophysiology and Emotion," New York: Rockefeller Univ. Press and Russell Sage foundation. Denenberg, V. H. (1969). In A. Ambrose (Ed.), "Stimulation in early infancy," New York: Academic Press. Denenberg, V. H., and Karas, G. G. (1960). Interactive effects of age and duration of infantile experience on adult learning. Psychol. Rep. 7, 313-322. Henderson, N. D. (1966). Changes in open-field behavior as a result of experimenter manipulation before or after intense shock. Can. J. Psychol. 20, 296-301. Hofer, M. A. (1970). Physiological responses of infant rats to separation from their mothers. Science 168,871-873. Levine, S. (1969). In A. Ambrose (Ed.), "Stimulation in Early Infancy," New York: Academic Press.
HANDLING AFTER EARLY WEANING
509
Levine, S., and Lewis, G. W. (1959). Critical periods for the effects of infantile experience on maturation of stress response. Science 129, 42-43. Levine, S., and Otis, C. S. (1958). The effects of handling before and after weaning on the resistance of albino rats to later deprivation. Can. J. Psychol. 12, 103-108. Meier, G. W., a n d Schutzman, L. H. (1968). Mother-infant interactions and experimenter manipulation: Confounding or misidentification. Develop. Psychobiol. 1, 141-145. Novgkovfi, V. (1966). Weaning of young rats: Effect of time on behavior. Science 151, 475-476. Nov~kov~t, V. (1970). Premature weaning of the rat: Its effect on the higher nervous activity and the chemical pattern of neurons. In S. Kazda and V. H. Denenberg (Eds.), "The Post-Natal Development of Phenotype," pp. 337-347. Academia: Prague. Russell, P. A.. (1971). Infantile stimulation in rodents: a consideration of possible mechanisms. Psychol. Bull. 75, 192-201. Russell, P. A., and Williams, D. I. (1973). Effects of repeated testing on rats locomotor activity in the open-field. Anirn. Behav. 21, 109-111. Schaeffer, T. (1963). Early "experience" and its effect on later behavioral processes in the rat. II. A critical factor in the early handling phenomenon. Trans. N.Y. Acad. ScL 25,871-889. Thoman, E. B., and Levine, S. (1970). Hormonal and behavioral changes in the rat mother as a function of early experience treatments of the offspring. PhysioL Behav. 5, 1417-1421. Thoman, E. B., and Arnold, W. S. (1968). Incubator rearing of infant rats without the mother: Effects on adult emotionality and learning. Develop. Psychol. 1,219-222. Whittier, J. L., and Littman, R. A. (1965). Very early weaning, weaning stimulation, and adult exploratory and avoidance behavior of the white rat. Can. J. Psychol. 19, 288-303. Weininger, O. (1953). Mortality of albino rats under stress as a function of early handling. Can. J. Psychol. 7, 111-114. Weininger, O. (1956). The effects of early experience on behavior and growth characteristics. J. Comp. PhysioL PsychoL 49, 1-9. Williams, D. I., and Russell, P. A. (1972). Open-field behaviour in rats: effects of handling, sex, and repeated testing. Brit. J. Psychol. 63, 593-596. Williams, D. I., and Wells, P. A. (1970). Differences in home cage emergence in relation to infantile handling. Psychon. ScL 18, 168-169.
BRIEF REPORT Handling Rat Pups After Early Weaning z DAVID I. WILLIAMS, GILLIAN B. BAILEY, and MICHAEL H. S. LEE
Department of Psychology, The University, Hull, England
To determine if handling infant rats acts directly to change development or whether the effect is an indirect one through the mother, pups were weaned at 14 days and the dam removed; half the fitters were handled on the next 8 days. Handling produced long-term changes in weight and behavior. Handling rat pups early in life produces gross physiological and behavioral changes both in development and in the adult animal (cf. Denenberg, 1967; Levine, 1969). The question arises as to whether this phenomenon is mediated by direct action of the stimulation on the neonate or through some indirect path (Russell, 1971). This latter situation would arise if the mother reacted to the treatment of her pups, and her subsequent behavior produced the long-term changes observed in the young (Meier and Schutzman, 1968). That there is a direct effect is not in doubt (Denenberg, 1969). But it is difficult to establish whether this momentary change results in the effects seen in the adult animal, for after stimulation the pup has to be returned to its mother and so is subject to her influence. The ideal solution is simple: remove the mother. In practice this is difficult as the mother is responsible for feeding and general care of her offspring. Artificial feeding methods (Thoman and Arnold, 1968) and early weaning techniques (Whittier and Littman, 1965) have been developed, but as these involve handling of the young they preclude a test of the infantile stimulation effect. In this study it has proved possible to use the early weaning procedure of Whittier and Littman without touching the pups in any way. So the mother was removed from the litter at 14 days after birth, 7 days before the normal weaning time. This enabled pups to be handled during the "sensitive" preweaning period without the mother, and so eliminate indirect maternal stimulation effects. 1This research was supported by grants from the Medical Research Council of Great Britain. 505 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
506
WILLIAMS, BAILEY AND LEE
Three days before the expected date of parturition 23 pregnant black hooded rats (strain PVG/C) were housed individually in opaque plastic boxes measuring 21 × 40.5 X 15 cm with wire mesh lids. The experimental room was illuminated for 16 hr per day and maintained at a temperature of 26 + I°C. At birth all litters were culled randomly to six pups and left undisturbed, except for replenishment of food and water, until Day 14 of life. On Day 14 mothers were removed and pups provided with a diet of brown wholemeal bread soaked in warm sterilized milk; water was always available and solid food (Oxoid diet 41B) was introduced on Day 19. Solid food only was provided from Day 21. Litters were allocated to either a handled (H) or nonhandled (NH) condition. The handling procedure involved removing the mother from the cage and taking each pup individually from the nest by hand and placing it in a separate compartment which measured 10 cm a in a wooden holding box; when all the pups had been removed they were replaced in the same manner; the operation took approximately 30 sec for each pup. Handling took place on days 14-21 inclusive; apart from this treatment no litter was disturbed until weighing at 25 days. At 53 + 2 days 60 rats with equal numbers of each sex from each group were tested in an open-field apparatus for three daily 3-min trims using the procedure of Williams and Russell (1972). Rats were selected evenly across litters but, because of random culling at birth giving different sex ratios within litters, numbers from each litter were not equal. The maximum number of animals from any one litter was two in each subgroup of 15; variation due to litter factors was thus small. Each rat was weighed before testing in the open field. Of the animals tested in the open-field 48 were assayed for adrenal ascorbic acid at 63 days of age using the procedure of Levine and Lewis (1959); half were subjected to a stress of three scrambled electric shocks (1.7 mA) of 30-sec duration delivered randomly in a 3-rain period prior to testing. Forty-two animals that had not been run in the open-field were tested on a standard cage-emergence procedure (Williams and Wells, 1970) at 76 days of age. Four complete litters (1H and 3NH) were destroyed prior to 21 aays as they appeared unlikely to survive. Results are presented from the remaining 19 litters (10H : 9NH); from these 4H and 6NH pups died prior to weaning. Total losses were, therefore, 10H and 24NH animals. At 25 days H animals were heavier than NH but the difference was not significant. By 50 days, however, males were significantly heavier than females (F(1,56) = 101.32, P < 0.001), and H animals significantly heavier than NH (F(1,56) = 16.14, P < 0.001). Ambulation scores in the open field showed a significant Trials X Sex × Treatment interaction (F (2,112)= 4.04, P < 0.05) which arose because H females did not show the drop-off in ambulation over time as did the other groups. The main interest here in the open-field measures lies in the index of entries into the center circle (Russell and
507
HANDLING AFTER EARLY WEANING TABLE 1 Open-Field Measuresa Entries into center
Defecation
Weight at 25 days
Weight at 50 days
Handled Male Female
35.5 3 41.87
2.31 1.38
38.96 38.07
157.87 125.20
Nonhandled Male Female
30.73 40.38
2.20 1.11
37.57 36.50
143.67 114.87
Ambulation
aEach figure is the mean over all trials. Weights are means expressed in grams.
Williams, 1973), where females made significantly more entries than males = 4.63, P < 0.05), and more importantly, H rats made significantly more entries than NH (F(1,56) = 17.71, P > 0 . 0 0 1 ) . Defecation scores showed the expected trend of males greater than females and NH greater than H but differences proved nonsignificant. Data on weight and open-field scores are shown in Table 1. On the emergence from cage measure (Rearing) a Mann-Whitney U test was used to analyze data separately for sex; H females (median 8.5 sec) reared faster than NH females (median 32 sec) (U = 37, nl = 10, n2 = 14, P < 0 . 0 5 ; 1-tailed), the male difference was in the same direction (H median 13.4 sec; NH median 30 sec) but this was not significant. For the adrenal ascorbic acid assay the mean for stressed animals was lower than for the control. While there were no significant differences between the groups in the control condition, there was in the stressed condition a significant Sex × Treatment interaction (F(1,20) = 6.81, P < 0.025). This arose because of a greater depletion of adrenal ascorbic acid in NH as compared with H females; this difference was not shown in males. The early weaning technique was successful in that the majority of pups survived. More NH than H animals were lost. This is contrary to findings in our own laboratory and elsewhere (Thoman and Levine, 1970) where, when handling is carried out with the mother present, H animals are more often lost. So differences due to handling cannot be explained on a "survival of the fittest" basis that handling selects stronger nonemotional animals by stressing and subsequently killing the weaker pups; although this hypothesis may well be relevant to some reported studies. Over-all then, as evidenced primarily by the weight measure and entries into the center of the open field, the handling treatment was effective in producing long-term changes. It is noteworthy that this procedure seemed to
(F(1,56)
508
WILLIAMS,BAILEY AND LEE
have more effect on females as shown by ambulation, rearing, and adrenal ascorbic acid depletion. These differences were found despite the fact that both groups would have been subject to stress in removing the mother at such an early date (Hofer, 1970). Also both groups would be equally liable to cool which presents problems for the hypothesis that infantile stimulation effects are mediated by hypothermia (Schaeffer, 1963). In addition, it is clear that this effect is more than habituation to handling influencing later behavioral tests (Henderson, 1966) for clear-cut physical differences in weight were found. A further problem is that an effect is obtained here with handling only in the third week postpartum. Other workers (Denenberg and Karas, 1960; Schaeffer, 1963) using the normal procedure of having the mother present failed to show an effect of handling during this period. It is tempting to speculate that in these studies the mother may have acted as a buffer to handling. But, on the other hand, there have been reports (Weininger, 1953; 1956) of handling being effective in the postweaning period, although these results have been generally discounted (Denenberg, 1964). Early weaning produces many behavioral and physiological differences which persist into adulthood (e.g., Nov~kova, 1966; 1970). The effectiveness of handling so late in the preweaning period could have resulted from an interaction with the effects of early weaning. Nevertheless the fact remains that a handling effect can be produced in the absence of the mother showing that there must be some direct effect of stimulation producing subsequent changes in development and adult behavior. This is not to say that when the mother is present her behavior is not relevant to the production of long-term changes resulting from handling; this indirect route could also be operative.
REFERENCES Denenberg, V. H. (1964). Critical periods, stimulus input, and emotional reactivity: A theory of infantile stimulation. Psychol. Rev. 71, 335-351. Denenberg, V. H. (1967). In D. C. Glass (Ed.), "Biology of Behavior: Neurophysiology and Emotion," New York: Rockefeller Univ. Press and Russell Sage foundation. Denenberg, V. H. (1969). In A. Ambrose (Ed.), "Stimulation in early infancy," New York: Academic Press. Denenberg, V. H., and Karas, G. G. (1960). Interactive effects of age and duration of infantile experience on adult learning. Psychol. Rep. 7, 313-322. Henderson, N. D. (1966). Changes in open-field behavior as a result of experimenter manipulation before or after intense shock. Can. J. Psychol. 20, 296-301. Hofer, M. A. (1970). Physiological responses of infant rats to separation from their mothers. Science 168,871-873. Levine, S. (1969). In A. Ambrose (Ed.), "Stimulation in Early Infancy," New York: Academic Press.
HANDLING AFTER EARLY WEANING
509
Levine, S., and Lewis, G. W. (1959). Critical periods for the effects of infantile experience on maturation of stress response. Science 129, 42-43. Levine, S., and Otis, C. S. (1958). The effects of handling before and after weaning on the resistance of albino rats to later deprivation. Can. J. Psychol. 12, 103-108. Meier, G. W., a n d Schutzman, L. H. (1968). Mother-infant interactions and experimenter manipulation: Confounding or misidentification. Develop. Psychobiol. 1, 141-145. Novgkovfi, V. (1966). Weaning of young rats: Effect of time on behavior. Science 151, 475-476. Nov~kov~t, V. (1970). Premature weaning of the rat: Its effect on the higher nervous activity and the chemical pattern of neurons. In S. Kazda and V. H. Denenberg (Eds.), "The Post-Natal Development of Phenotype," pp. 337-347. Academia: Prague. Russell, P. A.. (1971). Infantile stimulation in rodents: a consideration of possible mechanisms. Psychol. Bull. 75, 192-201. Russell, P. A., and Williams, D. I. (1973). Effects of repeated testing on rats locomotor activity in the open-field. Anirn. Behav. 21, 109-111. Schaeffer, T. (1963). Early "experience" and its effect on later behavioral processes in the rat. II. A critical factor in the early handling phenomenon. Trans. N.Y. Acad. ScL 25,871-889. Thoman, E. B., and Levine, S. (1970). Hormonal and behavioral changes in the rat mother as a function of early experience treatments of the offspring. PhysioL Behav. 5, 1417-1421. Thoman, E. B., and Arnold, W. S. (1968). Incubator rearing of infant rats without the mother: Effects on adult emotionality and learning. Develop. Psychol. 1,219-222. Whittier, J. L., and Littman, R. A. (1965). Very early weaning, weaning stimulation, and adult exploratory and avoidance behavior of the white rat. Can. J. Psychol. 19, 288-303. Weininger, O. (1953). Mortality of albino rats under stress as a function of early handling. Can. J. Psychol. 7, 111-114. Weininger, O. (1956). The effects of early experience on behavior and growth characteristics. J. Comp. PhysioL PsychoL 49, 1-9. Williams, D. I., and Russell, P. A. (1972). Open-field behaviour in rats: effects of handling, sex, and repeated testing. Brit. J. Psychol. 63, 593-596. Williams, D. I., and Wells, P. A. (1970). Differences in home cage emergence in relation to infantile handling. Psychon. ScL 18, 168-169.