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Maternal separation affects infant rats' behavior
BEHAVIORAL BIOLOGY, 9, 629-633 (1973), Abstract No. 3107
BRIEF REPORT Maternal Separation Affects Infant Rats' Behavior*
MYRON A. H O F E R
Department of Psychiatry, Albert Einstein College of Medicine at Montefiore Hospital, Bronx, New York 10467 Eig~ateen hour separations of 2 wk old Wistar rat pups from their mothers were found to increase levels of activity, elimination and selfgrooming in a novel environment and to delay sleep onset. A control group supplied with nonlactating foster mothers suggested that whereas the sleep onset changes could have a nutritional mechanism, the waking behavioral effects of separation probably depended on deprivation of behavioral interaction with the mother.
Although several studies have described behavioral changes in young primates soon after separation from their mother (Harlow and Zimmerman, 1959; Kaufman and Rosenblum, 1969; Hinde and Spencer-Boothe, 1971), there has been no evidence that more primitive mammals, such as tile rat, are similarly sensitive to this early experience. Furthermore, even in the primate studies, little information is available as to the importance of nutritional deprivation in determining the behavioral changes of the newly separated nursing infants. Previous studies from this laboratory have described autonomic cardiac rate changes in 2 wk old rat pups after short periods of maternal deprivation, which apparently have a nutritional mechanism involving ~-adrenergic pathways (Hofer, 1971; Hofer and Weiner, 1971). The following studies describe behavioral changes in 2 wk old rat pups separated from their mothers for 1 8 h r and evaluate the role o f nutritional deprivation in these effects. Three conditions were studied: normally mothered, separated and a group supplied with a nonlactating mother. This last group sustained the same nutritional deprivation as the separated pups but continued to have the opportunity for behavioral interaction with the mother. Twelve litters of a Wistar-derived strain (Carworth CFN) were born and maintained in our laboratory undisturbed until 2 wk of age on a reversed day-night cycle after being culled to 8-9 pups within the first 3 days of life. On the first day of *Supported by a Research Scientist Development Award and by Project Grant MH 16929 from the National Institute of Mental Health. I thank Harry Shah" and Hedda Orkin for assistance in the laboratory. 629 Copyright © 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
630
HOFER
each experiment, at 5 PM, the pups of 2 litters were divided so that 2 pups from each litter were distributed to each of the 3 experimental groups. Thermostatically regulated heat was supplied to the 4 separated pups through the solid composition b o t t o m of the cage. One of the mothers was lightly anesthetized with ether and her teats were ligated with 5-0 silk, she was then returned to her own cage, which was reduced to half its normal floor space. She could thus move only a few cm away from her 4 pups for the ensuing 18 hr. The final group of 4 pups remained with a normal mother until 0.5 hr prior to testing the following morning. All pups were individually marked with ink on their fur after the mothers had been removed 0.5 hr prior to testing. Since rat pups of this age are normally almost never alone, they were observed in groups of 4, for 10 min after being placed in a 30 × 20 cm plastic tray on an electronic activity platform (Lafayette Instruments) which transduces horizontal and vertical movements into counts registered each minute. In addition, observations were made 1 on self-grooming behavior, the occurrence of defecation or urination in a group (elimination), and sleep onset as judged by behavioral criteria for 'active' or 'REM' s l e e p - a b s e n t postural tonus accompanied by intermittent myoclonic twitching (Jouvet-Mounier, Astic and Locote, 1970). Axillary temperatures of all pups were recorded, without disturbing the pups, several minutes prior to testing. TABLE 1 Behavioral Measures After 18 hr Under Different Mothering Conditions (Each Condition Represented by 24 Subjects Studied in 6 Groups) Conditions Measures Activity count (median)
Normal mother
Nonlactating mother
Separated
1784
1458
Grooming No. pups No. episodes
9 14
14 21
20* 56*
Elimination No. groups No. episodes
1 1
0 0
4 7*
23** 2.8**
5 .8
1 .3
"Active sleep" No. pups median duration (min)
2595**
*,**Statistically different from other two conditions at P < 0 . 0 5 and P < .02, respectively, by Mann Whitney U tests. lIn an independent study inter-observer reliability was found to be 87% for self-grooming behavior, 100% for elimination and 96% for active sleep onset of pups observed together in groups of 4.
MATERNAL DEPRIVATION IN RATS
631
The results in Table 1 show that the separated pups were significantly more active than the other 2 groups on the three categories of waking behavior. The normally mothered pups showed by far the most rapid onset of behavioral evidence of active ('REM') sleep. In this category, the separated group were no different from those provided with a nonlactating foster mother. Analysis of variance of the time course of the activity platform counts (Fig. 1) showed that all three groups of pups tended to become less active 400 q,,.
, ./,a\ "~,...-a,-...,,._...A,,, -
lo0
I
i
i
i
i
I
TIME IN MINUTES
Fig. 1. Trends of activity counts over 10 min observation period for the 3 groups: (o) separated; (o) normal mother; (a) nonlactating mother.
over the 10 min in the novel environment ( P < 0.01). The separated pups had tile same initial level of activity as the two other groups but decreased their activity level less rapidly over the 10 min observation. There was a significant overall difference between the groups (P < 0.05) and a difference in the trend over time (group-time interaction; P < 0.05). This suggests a difference in behavioral habituation processes produced by the prior experiences of these pups. The foster-mothered and normally mothered groups showed almost identical patterns. The different behavioral measures have been plotted separately over time (Fig. 2). The differences between groups in self-grooming and elimination appear predominantly in the first half of the observation period while the difference in "active" sleep onset occurs mainly in the latter half. Body weight changes in the 3 groups over the 18 hr of the experiment showed the expected weight gain in the normally mothered pups (median + 2.8g; range + 1.5 to + 5.0g). Littermates in the other two conditions consistently lost weight. Those provided with the nonlactating foster mother lost significantly ( P < 0.01) more weight (median -2.3 g; range -1.3 to -3.7) than separated pups ( m e d i a n - 1 . 4 g ; r a n g e - 0 . 7 t o - 2 . 9 ) . Axillary temperatures were identical for separated and nonnutritive foster mother groups while normally mothered pups were 0.9°C warmer (P < 0.01). This experiment shows that rat pups which have been subjected to nutritional deprivation for 18 hr, but provided with the opportunity for behavioral interaction with a mother during this period, fail to show the
632
HOFER
SELF GROOMING
_
I- °
2
11F
ELIMINATION
Z
16
o
'ACTIVE' SLEEP
~
14
I
12
I
I0 8
,P',,. /
6i
P-""
I I I I
I
I
I
\b
"~
// ~.."~-'-~",.,,. O
2
3
4 5 6 7 TiME (Min)
8
9
10
Fig. 2. Number of pups showing individual behaviors each minute for the 3 groups: (o) separated; (e) normal mother; (zx) nonlactating mother.
hyperactivity characteristic of separated pups. One may infer from these results either (a) that absence of behavioral interaction with the mother rather than nutritional deficit is responsible for the behavioral effects or (b) that as a result of unsuccessful nursing attempts, the pups provided with a nonlactating mother were too exhausted for a nutritional mechanism to operate. The increased weight loss in the nonlactating mothered group may well result from evacuation of gastrointestinal tract contents resulting from specific stimulation by the mother. In addition, this group may have been stimulated to greater activity by the nonlactating mother than the separated group over the 18 hr period prior to testing. Recently completed experiments (Hofer, 1973) show that artificial feeding does not prevent the behavioral effects of separation and argues in favor of the first inference from the present results. To substantiate this hypothesis, it would be desirable to find out if provision of specific aspects of the mother-infant behavioral interaction are capable of preventing behavioral change due to maternal separation. The provision of warmth seems an unlikely candidate, since the behavioral differences found were in the
MATERNAL DEPRIVATION IN RATS
633
opposite direction from those which could be ascribed to body temperature
differences. It is not yet clear whether the level of tactile stimulation is the important factor, or whether specific behavioral interactions such as licking and grooming are crucial. Certainly, the differences in elimination during testing could reflect the previous elicitation of defecation and urination by the mother's licking the pups' anogenital region instead of reflecting differences in "emotionality." Alternately, the behavioral effects could depend on olfactory or vestibular stimulation. Possibly, all of these factors are involved, additively. The finding that behavioral evidences of paradoxical sleep are delayed in pups provided with a nonlactating foster mother to the same extent as after complete maternal deprivation suggests that separation from the mother at this age may affect this aspect of behavior through a nutritional (rather than social interaction) mechanism. Previous work has shown that nonlactating mothers fail to prevent the cardiac effects seen after separation (Hofer and Weiner, 1971) and cardiac rate has been subsequently found to have a nutritional regulatory mechanism at this age (Hofer, 1971). These sleep onset effects, however, may depend on some other differences between nonlactating and normal mothers. These experiments demonstrate that short periods of separation of 2 wk old rats from their mothers alter their state so that they respond to a novel environment with increased activity of several types and a lessened tendency to fall asleep. These alterations in level of behavioral arousal are reminiscent of the increased motor agitation and vocalization of recently separated monkey infants (Harlow and Zimmerman, 1959; Kaufman and Rosenblum, 1969; Hinde and Spencer-Boothe, 1971).
REFERENCES Harlow, H. F., and Zimmerman, R. R. (1959). Affectional responses in the infant monkey. Science 130, 421-432. Hinde, R. A., and Spencer-Boothe, Y. (1971). Effects of brief separation from mother on rhesus monkeys. Science 173, 111-118. Hofer, M. A. (1971). Cardiac rate regulated by nutritional factor in young rats. Science 172, 1039-1041. Hofer, M. A. (1973). The role of nutrition in the physiological and behavioral effects of maternal separation on two week old rat pups. Psychosom. Med. in press. Hofer, M. A., and Weiner, H. (1971). The development and mechanisms of cardiorespiratory responses to maternal deprivation in rat pups. Psychosom. Med. 33, 353-362. Jouvet-Mounier, D., Astic, L., and Locote, D. (1970). Ontogenesis of the state of sleep in rat, cat and guinea pig during the first postnatal month. Develop. Psychobiol. 2, 216-239. Kaufman, I. C., and Rosenblum, L. (1969). Effects of separation from mother on the emotional behavior of infant monkeys. Ann. N. Y. Acad. Sci. 159, 681-696.
BRIEF REPORT Maternal Separation Affects Infant Rats' Behavior*
MYRON A. H O F E R
Department of Psychiatry, Albert Einstein College of Medicine at Montefiore Hospital, Bronx, New York 10467 Eig~ateen hour separations of 2 wk old Wistar rat pups from their mothers were found to increase levels of activity, elimination and selfgrooming in a novel environment and to delay sleep onset. A control group supplied with nonlactating foster mothers suggested that whereas the sleep onset changes could have a nutritional mechanism, the waking behavioral effects of separation probably depended on deprivation of behavioral interaction with the mother.
Although several studies have described behavioral changes in young primates soon after separation from their mother (Harlow and Zimmerman, 1959; Kaufman and Rosenblum, 1969; Hinde and Spencer-Boothe, 1971), there has been no evidence that more primitive mammals, such as tile rat, are similarly sensitive to this early experience. Furthermore, even in the primate studies, little information is available as to the importance of nutritional deprivation in determining the behavioral changes of the newly separated nursing infants. Previous studies from this laboratory have described autonomic cardiac rate changes in 2 wk old rat pups after short periods of maternal deprivation, which apparently have a nutritional mechanism involving ~-adrenergic pathways (Hofer, 1971; Hofer and Weiner, 1971). The following studies describe behavioral changes in 2 wk old rat pups separated from their mothers for 1 8 h r and evaluate the role o f nutritional deprivation in these effects. Three conditions were studied: normally mothered, separated and a group supplied with a nonlactating mother. This last group sustained the same nutritional deprivation as the separated pups but continued to have the opportunity for behavioral interaction with the mother. Twelve litters of a Wistar-derived strain (Carworth CFN) were born and maintained in our laboratory undisturbed until 2 wk of age on a reversed day-night cycle after being culled to 8-9 pups within the first 3 days of life. On the first day of *Supported by a Research Scientist Development Award and by Project Grant MH 16929 from the National Institute of Mental Health. I thank Harry Shah" and Hedda Orkin for assistance in the laboratory. 629 Copyright © 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
630
HOFER
each experiment, at 5 PM, the pups of 2 litters were divided so that 2 pups from each litter were distributed to each of the 3 experimental groups. Thermostatically regulated heat was supplied to the 4 separated pups through the solid composition b o t t o m of the cage. One of the mothers was lightly anesthetized with ether and her teats were ligated with 5-0 silk, she was then returned to her own cage, which was reduced to half its normal floor space. She could thus move only a few cm away from her 4 pups for the ensuing 18 hr. The final group of 4 pups remained with a normal mother until 0.5 hr prior to testing the following morning. All pups were individually marked with ink on their fur after the mothers had been removed 0.5 hr prior to testing. Since rat pups of this age are normally almost never alone, they were observed in groups of 4, for 10 min after being placed in a 30 × 20 cm plastic tray on an electronic activity platform (Lafayette Instruments) which transduces horizontal and vertical movements into counts registered each minute. In addition, observations were made 1 on self-grooming behavior, the occurrence of defecation or urination in a group (elimination), and sleep onset as judged by behavioral criteria for 'active' or 'REM' s l e e p - a b s e n t postural tonus accompanied by intermittent myoclonic twitching (Jouvet-Mounier, Astic and Locote, 1970). Axillary temperatures of all pups were recorded, without disturbing the pups, several minutes prior to testing. TABLE 1 Behavioral Measures After 18 hr Under Different Mothering Conditions (Each Condition Represented by 24 Subjects Studied in 6 Groups) Conditions Measures Activity count (median)
Normal mother
Nonlactating mother
Separated
1784
1458
Grooming No. pups No. episodes
9 14
14 21
20* 56*
Elimination No. groups No. episodes
1 1
0 0
4 7*
23** 2.8**
5 .8
1 .3
"Active sleep" No. pups median duration (min)
2595**
*,**Statistically different from other two conditions at P < 0 . 0 5 and P < .02, respectively, by Mann Whitney U tests. lIn an independent study inter-observer reliability was found to be 87% for self-grooming behavior, 100% for elimination and 96% for active sleep onset of pups observed together in groups of 4.
MATERNAL DEPRIVATION IN RATS
631
The results in Table 1 show that the separated pups were significantly more active than the other 2 groups on the three categories of waking behavior. The normally mothered pups showed by far the most rapid onset of behavioral evidence of active ('REM') sleep. In this category, the separated group were no different from those provided with a nonlactating foster mother. Analysis of variance of the time course of the activity platform counts (Fig. 1) showed that all three groups of pups tended to become less active 400 q,,.
, ./,a\ "~,...-a,-...,,._...A,,, -
lo0
I
i
i
i
i
I
TIME IN MINUTES
Fig. 1. Trends of activity counts over 10 min observation period for the 3 groups: (o) separated; (o) normal mother; (a) nonlactating mother.
over the 10 min in the novel environment ( P < 0.01). The separated pups had tile same initial level of activity as the two other groups but decreased their activity level less rapidly over the 10 min observation. There was a significant overall difference between the groups (P < 0.05) and a difference in the trend over time (group-time interaction; P < 0.05). This suggests a difference in behavioral habituation processes produced by the prior experiences of these pups. The foster-mothered and normally mothered groups showed almost identical patterns. The different behavioral measures have been plotted separately over time (Fig. 2). The differences between groups in self-grooming and elimination appear predominantly in the first half of the observation period while the difference in "active" sleep onset occurs mainly in the latter half. Body weight changes in the 3 groups over the 18 hr of the experiment showed the expected weight gain in the normally mothered pups (median + 2.8g; range + 1.5 to + 5.0g). Littermates in the other two conditions consistently lost weight. Those provided with the nonlactating foster mother lost significantly ( P < 0.01) more weight (median -2.3 g; range -1.3 to -3.7) than separated pups ( m e d i a n - 1 . 4 g ; r a n g e - 0 . 7 t o - 2 . 9 ) . Axillary temperatures were identical for separated and nonnutritive foster mother groups while normally mothered pups were 0.9°C warmer (P < 0.01). This experiment shows that rat pups which have been subjected to nutritional deprivation for 18 hr, but provided with the opportunity for behavioral interaction with a mother during this period, fail to show the
632
HOFER
SELF GROOMING
_
I- °
2
11F
ELIMINATION
Z
16
o
'ACTIVE' SLEEP
~
14
I
12
I
I0 8
,P',,. /
6i
P-""
I I I I
I
I
I
\b
"~
// ~.."~-'-~",.,,. O
2
3
4 5 6 7 TiME (Min)
8
9
10
Fig. 2. Number of pups showing individual behaviors each minute for the 3 groups: (o) separated; (e) normal mother; (zx) nonlactating mother.
hyperactivity characteristic of separated pups. One may infer from these results either (a) that absence of behavioral interaction with the mother rather than nutritional deficit is responsible for the behavioral effects or (b) that as a result of unsuccessful nursing attempts, the pups provided with a nonlactating mother were too exhausted for a nutritional mechanism to operate. The increased weight loss in the nonlactating mothered group may well result from evacuation of gastrointestinal tract contents resulting from specific stimulation by the mother. In addition, this group may have been stimulated to greater activity by the nonlactating mother than the separated group over the 18 hr period prior to testing. Recently completed experiments (Hofer, 1973) show that artificial feeding does not prevent the behavioral effects of separation and argues in favor of the first inference from the present results. To substantiate this hypothesis, it would be desirable to find out if provision of specific aspects of the mother-infant behavioral interaction are capable of preventing behavioral change due to maternal separation. The provision of warmth seems an unlikely candidate, since the behavioral differences found were in the
MATERNAL DEPRIVATION IN RATS
633
opposite direction from those which could be ascribed to body temperature
differences. It is not yet clear whether the level of tactile stimulation is the important factor, or whether specific behavioral interactions such as licking and grooming are crucial. Certainly, the differences in elimination during testing could reflect the previous elicitation of defecation and urination by the mother's licking the pups' anogenital region instead of reflecting differences in "emotionality." Alternately, the behavioral effects could depend on olfactory or vestibular stimulation. Possibly, all of these factors are involved, additively. The finding that behavioral evidences of paradoxical sleep are delayed in pups provided with a nonlactating foster mother to the same extent as after complete maternal deprivation suggests that separation from the mother at this age may affect this aspect of behavior through a nutritional (rather than social interaction) mechanism. Previous work has shown that nonlactating mothers fail to prevent the cardiac effects seen after separation (Hofer and Weiner, 1971) and cardiac rate has been subsequently found to have a nutritional regulatory mechanism at this age (Hofer, 1971). These sleep onset effects, however, may depend on some other differences between nonlactating and normal mothers. These experiments demonstrate that short periods of separation of 2 wk old rats from their mothers alter their state so that they respond to a novel environment with increased activity of several types and a lessened tendency to fall asleep. These alterations in level of behavioral arousal are reminiscent of the increased motor agitation and vocalization of recently separated monkey infants (Harlow and Zimmerman, 1959; Kaufman and Rosenblum, 1969; Hinde and Spencer-Boothe, 1971).
REFERENCES Harlow, H. F., and Zimmerman, R. R. (1959). Affectional responses in the infant monkey. Science 130, 421-432. Hinde, R. A., and Spencer-Boothe, Y. (1971). Effects of brief separation from mother on rhesus monkeys. Science 173, 111-118. Hofer, M. A. (1971). Cardiac rate regulated by nutritional factor in young rats. Science 172, 1039-1041. Hofer, M. A. (1973). The role of nutrition in the physiological and behavioral effects of maternal separation on two week old rat pups. Psychosom. Med. in press. Hofer, M. A., and Weiner, H. (1971). The development and mechanisms of cardiorespiratory responses to maternal deprivation in rat pups. Psychosom. Med. 33, 353-362. Jouvet-Mounier, D., Astic, L., and Locote, D. (1970). Ontogenesis of the state of sleep in rat, cat and guinea pig during the first postnatal month. Develop. Psychobiol. 2, 216-239. Kaufman, I. C., and Rosenblum, L. (1969). Effects of separation from mother on the emotional behavior of infant monkeys. Ann. N. Y. Acad. Sci. 159, 681-696.