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Maternal and nutritional factors in maintenance of infant rat cardiac rate following maternal separation
Physiology & Behavior, Vol. 16, pp. 521-527. Pergamon Press and Brain Research Publ., 1976. Printed in the U.S.A.
Maternal and Nutritional Factors in Maintenance of Infant Rat Cardiac Rate Following Maternal Separatioff MANFERD D. KOCH 2 AND WILLIAM J. ARNOLD
Department o f Psychology, University o f Nebraska-Lincoln, Lincoln, ArE 68508 (Received 20 March 1975) KOCH, M. D. AND W. J. ARNOLD. Maternal and nutritional factors in maintenance of infant rat cardiac rate following maternal separation. PHYSIOL. BEHAV. 16(5) 521-527, 1976. - Nutrient intake frequency and the presence of a surrogate mother were studied as factors influencing preweanling rat heart rates following maternal separation. Three groups of pups were separated from lactating females at 4, 7, and 10 days of age and maintained for 16 hr under the conditions of high frequency nutrient intake, low frequency nutrient intake, sham fed with no nutrient intake, or both high frequency intake and presence of a foster mother. A group of 14-day-olds were tested under the first three nutrient intake conditions. Results indicated that both nutritional factors and presence and behavior of the mother are influential in maintaininghigh sympathetic tone in preweanling rats. Maternal separation
Heart rate
Sympathetic
Parasympathetic
Frequency of nutrient intake
normal basal heart rate levels. Through the process of disproving alternative hypotheses, Hofer and Weiner conclude that the aspect of deprivation which is responsible for the decelatory heart rate effect may lie within the feeding interaction. However, the possibility still exists that some combination for proper nutrient intake and presence of adequate maternal stimuli are necessary for optimal heart rate maintenance. The initial purpose of the present experiment was to determine whether frequency of nutrient intake is a crucial factor for maintaining high sympathetic tone and cardiac rate prior to weaning. Mother rats repeat a cycle of nursing and moving away from the pups throughout the rearing period prior to weaning [ 1]. Between 12 and 19 days of age the median nursing phase duration is 26 min and the mother is away from the pups for 54 min on the average [5]. Therefore, the periods between nutrient intake are of about 1 hr duration. In the present study it was hypothesized that frequencies of nutrient intake which approximate those occurring naturally, i.e., 1 per hr, would be effective in maintaining high resting heart rate levels, and that relatively low frequencies of nutrient intake which were used by Hofer [6], i.e., 1 feeding every 4 b_r, would result in decreasing heart rates following maternal separation although the same volume of formula was intubated over a 16 hr period. The second purpose of this experiment was to determine whether stimuli provided by the physical presence of a surrogate mother would be effective in elevating inactive heart rates under conditions where actual
STUDIES involving maternal deprivation have shown that depriving an infant of contact with its biological mother or adequate maternal substitute has deleterious effects on the offspring's ability to cope with stressful situations later in life [2, 8, 9, 10, 11]. Research is now beginning to show how specific physiological systems, such as the autonomic nervous system, may be directly altered by early maternal separation. Hofer [3] demonstrated that separating 14day-old rat pups from their natural mothers caused a decrease in the pups' active and inactive heart rates by 40% during the first 12 to 16 hr following separation. Although nutritional factors may be important, merely providing adequate nutrient intake to produce weight gains did not reverse bradycardia following maternal separation in this study. Hofer [4] reported a subsequent study which showed that reversal of the decrease in cardiac rate following 16 hr of maternal separation in 15-day-old rat pups is a function of the amount of milk intubated. The implication of this study is that cardiac rate appears to be controlled, at least in part, by quantitative and scheduling characteristics of nutrient intake. Hofer and Weiner [7] found no significant differences in heart rate functioning between pups housed with nonnursing mothers and those completely deprived of maternal contact. They conclude that the physiological changes resulting from maternal deprivation are not dependent on loss of the physical presence of the mother per se. These findings initially appear to diminish the likelihood that olfactory, auditory and tactile stimuli are essential for the maintenance of
aThis research was supported by Research Grants MH-17377-03 and MH-17377-04 from the National Institute of Mental Health, United States Public Health Service. 2Requests for reprints may be sent to Manferd D. Koch, Department of Psychology, University of Nebraska-Lincoln, Lincoln NE 68508. 521
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KOCH AND ARNOLD
nursing was precluded but pups were provided with normal occurring frequencies of nutrient intake. A total of three age levels, 4-, 7-, and 10-day-olds, were studied under each of the following conditions: (a) High Frequency (HF) group - animals tube fed bovine formula every hour following separation from their natural mothers; (b) Low Frequency (LF) group - animals tube fed once every 4th hour following separation and sham fed once every other hour; (c) Sham Fed (SF) group - animals sham fed only once very hour following sepataion; and (d) Foster Mother (FM) group - animals tube fed every hour following separation and housed with a foster mother whose teats had been surgically excised to preclude nursing. An additional group of 14-day-old pups was tested in an attempt to replicate in part Hofer's 1970 study in terms of the influence of nutritional factors on heart rate at that age. METHOD Animals and Surgery Twenty-five time-pregnant random-bred females with known dates of sperm positivity were obtained from the Charles River Company. During the experiment the females were housed in individual cages on ad lib food and water. Of these females, 24 delivered within 12 hr. Twenty-four hr prior to separating the pups from their mothers for testing, each pup was removed from the nest and two electrodes were implanted subcutaneously under light ether anesthesia. The electrodes consisted of fine 36 ga stainless steel wires which were implanted and formed into small loops with twisted ends. One electrode was positioned dorsally at right angles to the spine posterior to the forelegs and the second dorsally at right angles to the spine directly over the hind legs. Collodion was applied around the skin areas where the electrodes emerged to prevent recording artifacts due to electrode movement. The electrode implanting procedure took approximately 3 min; immediately after which the pups were returned to their home nests. Starting with litters born first, 40 pups were drawn from the total pool of male pups in the first 8 litters and were randomly assigned to the four experimental conditions at 4 days of age. The procedure of assigning pups to age levels on the basis of birth order was continued for age levels 7 and 10. The HF, LF, SF, and FM groups sizes for the three age levels were as follows: 4 days, n = 10; 7 days, n = 1 0 ; a n d 10 days, n = 11. This resulted in 124 pups being used. Animals were not disturbed between parturition and testing except for electrode implantation. A female which had previously reared two litters, the last of which was weaned 60 days prior to this study, was used as the foster mother. Thirty days prior to this experiment the female was placed under ether anesthesia and her teats were surgically excised to preclude nursing. Apparatus and Procedure When the pups reached their respective test ages, they were removed from their home cages and mothers, weighed, and marked for identification purposes. Since pups younger than 16 days are not homeothermic and body temperature is known to affect the cardiac intrinsic pacemaker [6], pups in the HF, LF, and SF groups were placed in incubators (20 x 26 x 15 cm) which were kept at a constant temperature between 32°C and 33°C. Maintenance of this temperature has been found to be of the
greatest importance for successful rearing of rat pups in the absence of a nesting female [12]. The temperature of the pups in the FM groups was maintained by the body heat from the foster mother; she crouched over the pups for long periods of time as if she were nursing them. Room temperature was maintained between 25°C and 27°C, and humidity was kept at 45%. The HF, LF and FM groups were fed a bovine milk formula consisting of 200 ml condensed milk, 200 ml distilled water, 40 g of instant dry milk, 2 cc Vi-Syneral, and the contents of one capsule of commercially-produced Methischol. The milk formula differs from that previously used by the investigators [8] in both water and instant dry milk content. The composition of the formula and volumes intubated at each feeding were based upon amounts which have previously been found sufficient to produce weight gains in long-term separated rat pups. Volumes of formula fed the three experimental groups at the three age levels are given in Table 1. (These volumes were prorated so that the groups of equal age received equal amounts of nutrient during the 16 hr of separation.) Pups of the FM group were fed the milk formula at rates and volumes identical to the HF group pups at the same age level. A stomach catheter, made of Silastic medical grade tubing (0.012 in I.D. and 0.025 in O.D.), reinforced with an inserted stainless steel suture wire, was used to intubate the formula at each feeding. The feeding process used involved inserting a feeding tube through the mouth into the stomach and injecting the appropriate volumes of formula via a syringe. The shamfeeding process used in both the SF and LF groups involved inserting a feeding tube through the mouth into the stomach of the pup for a period equal to that used for actual tube feeding in the HF, LF, and FM groups; however, no milk was injected. The sham-feeding process served as a control to equate handling and oral stimulation across the treatment groups. TABLE 1 VOLUMES OF BOVINE FORMULA FED ANIMALS IN EACH OF THREE EXPERIMENTALGROUPSAT FOUR AGE LEVELS Age in Days
HF
Experimental groups LF
FM
4 7 10 14
0.15 cc/hr 0.20 cc/hr 0.30 cc/hr 0.40 cc/hr
0.60 cc/4 hr 0.80 cc/4 hr 1.20 cc/4 hr 1.60 cc/4 hr
0.15 cc/hr 0.20 cc/hr 0.30 cc/hr --
Heart-rate recordings were taken at 0, 8, and 16 hr following separation from the mother using a Beckman Dynograph Recorder (Type RS) and a cardiotachometer coupler (Type 9857B). Lightweight recording clips were attached to the twisted ends of the implanted electrodes with a minimum amount of disturbance to the pup. The foster mother was gently removed from the nest just prior to each recording session and returned immediately after all measures were taken on FM group pups. Any movement on the part of the pup being recorded was noted on the chart paper and heart rates during periods of activity were not used. The pups were allowed approximately 30 sec to adapt to the placement of the recording clips prior to any recording. If a pup did not adapt, the clips were removed
MATERNAL SEPARATION AND HEART RATE
523 TABLE 2
MEAN BODY WEIGHTSAT SEPARATIONFROM THE MOTHERSAND AFTER 16 HR OF SEPARATION FOR ALL GROUPSSTUDIEDAT FOUR AGE LEVELS
Age in days 4 7 10 14
Mean Weights in Grams (S.D.) LF group SF group 0 hr 16 hr 0 hr 16 hr
HF group 0 hr 16 hr 11.9(1.0) 16.2(1.3) 22.3(2.1) 33.8(3.2)
14.2(1.1) 18.0(1.3) 24.9(2.0) 36.7(3.1)
11.9(1.1) 16.4(1.3) 21.9(2.0) 34.0(3.3)
14.0(1.1) 18.6(1.3) 24.5(1.9) 37.0(3.1)
and recordings were taken after the other pups had been recorded. Inactive heart rates during continuous 30 sec periods of recording constituted the measure of the dependent variable for subjects under all conditions. Recordings were always taken prior to the next feeding. RESULTS
Four-Day-OM Pups Mean body weights of 4-day-old pups when separation from the mother and after 16 hr of separation are given in Table 2. No significant difference among pup weights at the time of separation was found using a one way analysis of variance. However, a one way analysis of covariance of pups' weights after separation, using weights at the time of separation as the covariate, indicated a highly significant difference among groups, F(3,35) = 341.86, p<0.001. The
12.0(1.1) 16.3(1.4) 21.1(2.3) 33.4(2.8)
FM group 0 hr 16 hr
11.3(1.0) 11.7(0.9) 12.5(0.8) 15.6(1.3) 16.2(1.6) 17.3(1.4) 20.1(2.2) 22.6(2.0) 23.8(1.8) 31.4(2.6) ---
SF animals lost weight over the 16 hr of separation, and the groups which were fed gained weight. Animals housed with a foster mother did not gain as much weight as those in the other two groups, although they were fed identical quantities of milk. The lower weight gains of the FM group may be the result of greater activity induced by the presence and behavior of the foster mother, greater elimination due to the mother stimulating the anogenital area, altered autonomic nervous system functioning, or a combination of these three factors. Mean resting heart rates for the four groups at 0, 8, and 16 hr following separation from the mother are given in Fig. 1. A one way analysis of variance of group heart rates at 0 hr only approached significance, F(3,39) = 2.11, p>0.05. Because these initial heart rate means among groups were not equal, a 4 x 2 analysis of covariance was used to test for significance of group differences at 8 and 16 hr following separation. The overall
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FIG. 1. Cardiac responses of 4-day-old rat pups following separation from their natural mothers.
524 analysis of covariance on resting heart rates, using rates at 0 hr as the covariant, indicated that the differences among the groups were highly significant, F(3,36) = 17.79, p<0.005. A 4 × 2 analysis of variance indicated that the Hours and Groups × Hours factors were not significant. Orthogonal comparisons of covariance adjusted group heart rates over 8 and 16 hr indicated that the LF group had significantly higher mean rates than the HF group, F(1,36) = 9.11, p<0.005. Additional orthogonal comparisons indicated that the mean heart rates of the FM groups were significantly higher than the average of the two other groups which were fed (HF and LF), F(1,36) = 12.09, p<0.005. The average mean heart rates for the HF, LF, and FM groups was significantly higher than the mean heart rate of the SF group, F(1,36) = 25.90, p<0.005. The results did not support the hypothesis that normal occurring frequency of feeding was an important variable in maintenance of high sympathetic tone. However, contrary to results reported by Hofer [3], whether or not the pups were fed following separation did influence cardiac rate. The nutrient factor appears to be effected in maintaining sympathetic activity above that for pups totally deprived of food under conditions of long-term separation. AdditionaUy, the results indicated that stimuli associated with the presence of a maternal surrogate were sufficient to accelerate heart rates above levels occurring in that group shortly after separation from the natural mother and to increase heart rates significantly above those merely fed. Stimuli associated with the physical presence and behavior of the mother appear to be essential for the maintenance of high sympathetic tone. The foster mother may have provided more stimulation in attempting to nurse the pups than would have occurred under normal conditions. Because her nursing behavior was not regulated by milk release and uptake, she may have actually spent more time attempting to nurse than a lactating and nursing mother. Other research suggests that the total amount of time nonnutritive mothers spend with their pups may be higher than normal and that the rhythmic o n - o f f nursing pattern may be disrupted [7]. The possibility exists that in the FM condition an interaction effect between nutrient intake and stimuli of the surrogate mother accounts for a portion of the increases sympathetic tone and lower weight gains. Maternal stimuli, in the presence of nutriment, may have initially resulted in an increase in sympathetic discharge, achiorhydria and resultant decreased gastric and intestinal motility. The subsequent hourly intubations of nutriment, under conditions of diminished digestion, may have produced a greater arousal effect resulting in additionally increased sympathetic activity and tachycardia. The consequence of diminished digestion would be lower weight gains, as was observed in the FM pups.
Seven-Day-Old Pups Mean body weights among the four experimental groups over the 16 hr of separation showed the same general trends as were found for 4-day-old pups under similar rearing conditions (see Table 2). Again, no significant differences were found among group pup weights at 0 hr. Mean changes in body weights showed that FM pups gained less weight than HF or LF pups and that SF pups lost weight during the 16 hr period of separation. A one way analysis of covariance, using weights at the time of separation as the covariate, indicated a highly significant difference among
KOCH AND ARNOLD these group weights, F(3,35) = 260.44, p<0.001. Heart rate patterns similar to those of 4-day-olds were found for 7-day-old rat pups during the period of separation (see Fig. 2). Group heart rates at 0 hr did not differ significantly, but a 4 x 2 analysis of covariance of heart rates at 8 and 16 hr indicated that differences among the four groups were significant, F(3,36) --- 48.79, p<0.005. A 4 x 2 analysis of variance indicated that the Hours factor was not significant, although a significant Group x Hours factor was found, F(3,35) = 5.08, p<0.01, reflecting the rate increase in the HF and LF groups and the rate decrease in the SF group between 8 and 16 hr. Orthogonal comparisons of covariance adjusted group heart rates indicated that the difference between the HF and LF groups was not significant. Heart rate means of the FM group were significantly higher than the average of the HF and LF group means, F(1,36) = 38.42, p<0.005, again indicating that presence of maternal surrogate factors increases heart rates above those which occur when pups are simply provided adequate nutrient. The average of mean heart rates for all groups which were fed was significantly greater than the mean of the SF group, F(1,36) = 64.32, p<0.005, again implicating nutritional factors as important in maintaining heart rate.
Ten-Day-Old Pups The results of testing 10-day-old pups generally confirmed the results of 4- and 7-day-old pups reared under similar conditions. No significant differences existed among group pup weights at 0 hr. FM subjects gained less weight than HF or LF animals and SF animals lost weight during the period of separation (see Table 2). A one way analysis of covariance, using weights at the time of separation as the covariate, indicated a highly significant difference among the group weights after 16 hr of separation, F(3,39) = 530.18, p<0.001. Heart rate patterns, similar to those previously described for the other age levels studied, were found for 1-day-old pups (see Fig. 3). Group heart rates at 0 hr did not differ significantly, but a 4 x 2 analysis of covariance on resting heart rates at 8 and 16 hr indicated that the differences among the four groups were significant, F(,3,40) = 48.83, p<0.005. A 4 × 2 analysis of variance showed that the Groups x Hours interaction was significant, F(3,39) = 12.83, p<0.005, reflecting a decrease over hours for all groups except the FM group which increased. The Hours factor was not significant. Orghogonal comparisons of covariance adjusted group heart rates indicated a nonsignificant difference between the HF and LF groups, a significant difference between the combined HF and LF groups as contrasted to the FM groups, F(1,40) = 51.50, p<0.005, and a significant difference between the average of heart rate means of the fed groups contrasted to the heart rate mean of the SF group, F ( 1 , 4 0 ) = 51.35, p<0.005.
Fourteen-Day-Old Pups Hofer [3] reported that under the experimental conditions he used, physiological changes of 14-day-old pups following separation from the mother were not dependent upon starvation and dehydration. Because results of the current study indicated that nutrient intake was effective in maintaining cardiac rates at the 4-, 7-, and 10-day levels, a second segment of this study was conducted to determine if the nutritional factor was important at the 14-day-old level
MATERNAL SEPARATION AND HEART RATE
525
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H F GROUe :
280-
~'~'m
:
I. F GROUP o - . . . . o S F GROUP mm-. . . . . -m F M GROUP ±
1
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6
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16.5
HOURS AFTER SEPARATION
FIG. 2. Cardiac responses of 7-day-old rat pups following separation from their natural mothers.
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280. GROUI~ o-. . . . - o S F GROUP m-. . . . . • F M GROUP ±
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FIG. 3. Cardiac responses of 10-day-old rat pups following separation from their natural mothers.
526
KOCH AND ARNOLD
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FIG. 4. Cardiac responses of 14-day-old rat pups following separation from their natural mothers. under the experimental conditions employed in this study. Three groups were studied: (a) high frequency; (b) low frequency; and (c) sham fed. Volumes of formula intubated in the HF and LF groups are given in Table 1. Mean body weight at separation and after 16 hr are given in Table 2. A one way analysis of variance indicated no significant difference among pup weights at 0 hr. An analysis of covariance of weights at 16 hr, using weights at the time of separation as the covariate, indicated a highly significant difference among the group weights, F(2,23) = 117.18, p<0.001, although there was only a small difference between the mean weights of the groups which were fed. Mean heart rates for the three groups are given in Table 4. A one way analysis of variance of group heart rates at 0 hr only approached significance, F(2,24)-3.35, p>0.05. An analysis of covariance, using 0-hr heart rates as the covariate, indicated a significant overall difference at 8 and 16 hr, F(,2,24) = 8.65, p<0.005. The mean resting heart rates for the three groups decreased significantly over the 16 hr o f separation, F(1,23) = 23.47, p<0.005, although there was no significant Group × Hours interaction. The difference between the average of heart rate means for the HF and LF groups and the mean heart rate for the SF group was significant, F(1,24) = 12.52, p<0.005; the HF and LF did not differ significantly. These results are consistent with those of the other three age levels in showing that nutritional factors which prevent dehydration and starvation are important variables in the maintenance of cardiac rate in rat pups. DISCUSSION
Results of this study implicate at least two groups of
factors as critical in the maintenance of high sympathetic tone in rat pups prior to weaning: (a) nutritional factors, and (b) maternal stimuli. The first group of factors, nutritional, has at least a minimal influence on maintenance of heart rate levels. At each age level studied, heart rates for the groups artificially provided the milk formula were higher than for those merely sham fed. These results differ from those previously published by Hofer [3] in that he found no significant differences between the mean heart rates of 14-day-old pups which were fed every 4 hr and the heart rates of pups merely sham fed. Differences exist between the composition and volumes of milk formulae used in the two studies which could account for the different results. Hofer reportedly fed his pups nondiluted, reconstituted condensed milk which was considered to be sufficient to prevent starvation and dehydration, and it did produce weight gains. However, since condensed milk is slightly higher in dietary osmolarity (approximately 986 mosmols/1) than the formula used in the present study (approximately 901 mosmols/1), the former may have been less effective in maintaining heart rates because it was more dehydrating. Dehydration has been shown to reduce survival of neonatal rats [13]. A more marked difference which may explain the discrepant results between the earlier study and this research is the volume of formula fed during the period of separation. Hofer [ 3 ] fed each pup 0.8 ml of formula every 4 hr while in the present study twice that volume was fed - 1.6 cc/4 hr. The larger volumes may have been more effective in stimulating sympathetic activity since larger volumes ( 1 . 0 - 1 . 5 ml) have been shown to be more effective than smaller volumes (0.5 ml) in stimulating tachycardia in 14-day-old rat pups separated
MATERNAL SEPARATION AND H E A R T RATE
527
from their mothers for 16 hr prior to intubation [4]. The question of whether frequency of nutrient intake influences cardiac maintenance remains unanswered. Under the artificial feeding conditions used in this study, there appears to be little reliable difference between intubating formula once every 4 hr or once every hour. The only age level at which a significant difference occurred between the HF and LF condition was at 4 days of age, and that difference was in the direction opposite to that predicted, but no explanation is readily available for this unexpected difference. Hofer and Weiner [7] found that the mere presence of a nonlactating mother failed to prevent a decrease in heart rates over a 24 hr period. However, no attempt was made by the researchers to provide the pups with nutriment during the experiment. Hofer and Weiner hypothesized that it is possibly some aspect o f the feeding interaction with the mother that regulates sympathetic tone at high levels prior to weaning. Results of the present study suggest that high sympathetic tone is dependent on a combination of at least two factors, nutritional and maternal stimuli, but not necessarily nursing per se. The second factor, maternal stimuli, involves a complex of stimuli associated with the physical presence and behavior of the mother (or adequate maternal surrogate). Research has shown that heart rates occurring immediately following separation are somewhat depressed as compared with preseparation levels [6]. However in the present study when both factors, surrogate maternal stimuli and nutriment, were present, heart rates of pups at 4, 7, and 10 days of age appeared to accelerate toward what presumably were preseparation levels. The individual presence o f one of these two factors does not appear to be a sufficient stimulus to maintain sympathetic tone. These results suggest that in addition to the role of nutrition, maternal stimuli, such as stimulation of mic-
turation and defecation, physical contact, and possibly pheromones may be individually or in combination important in the regulation of autonomic functioning. It has been shown that rat pups as young as 2 days of age are able to behaviorally respond to maternal odors [ 14,15 ] which suggests the possibility that the FM pups in the present study may have been responding to olfactory cues associated with the mature female. Another possibility is that rat pups become conditioned to a total complex of stimuli associated with the adult female, and that these stimuli are capable of eliciting high sympathetic activity. One implication o f the findings of this study is that when the mother leaves the nest and is absent for a long period of time, which she must be in order to hunt for food under natural conditions, some physiological processes of the pups reverse. The result is that parasympathetic activity predominates causing a resultant lowering o f heart rate. This would be adaptive in decreasing activity and conserving energy. The longer the separation, the more marked the parasympathetic activity and the greater the resultant energy conservation. When the female returns to the nest and again initiates nursing, sympathetic activity would begin to predominate. A system of reciprocal, environmentally elicited autonomic responses would have high survival value for the species because high sympathetic activity should facilitate behaviors such as nipple-seeking and nursing, while parasympathetic activity would reduce random movement away from the safety of the nest. Another implication of these findings is the possibility that separating rat pups from their mothers for several days would affect the development of adult autonomic functioning. This is substantiated in part by Koch and Arnold's [8] finding that maternally deprived rats, as adults, showed aberrant autonomic reactivity to environmental stimuli as contrasted to rats reared with mothers.
REFERENCES 1. Grota, L. J. and R. Ader. Continuous recording of maternal behavior in Rattus Norvegicus. Anirrt Behav. 17" 722-729, 1969. 2. Harlow, H. F. and M. K. Harlow. Social deprivation in monkeys. Scient. Am. 207: 136-146, 1962. 3. Hofer, M. A. Physiological responses of infant rats to separation from their mothers. Science 168: 871-873, 1970. 4. Hofer, M. A. Cardiac rate regulated by nutritional factors in young rats. Science 172: 1039-1041, 1971. 5. Hofer, M. A. and M. Grabie. Cardiorespiratory regulation and activity patterns of rat pups studied with their mothers during the nursing cycle. Devel. Psychobiol. 4: 169-180, 1971. 6. Hofer, M. A. and M. F. Reiser. The development of cardiac rate regulation in preweanling rats. Psychosonz Med. 31: 372-388, 1969. 7. Hofer, M. A. and H. Weiner. Development and mechanisms of cardio-respiratory responses to maternal deprivation in rat pups. Psychosom. Med. 33: 353-362, 1971. 8. Koch, M. D. and W. J. Arnold. Effects of early social deprivation on emotionality in rats. J. cornp, physiol. Psychol. 78: 391-399, 1972.
9. Mason, W. A. and P. C. Green. The effects of social restriction on the behavior of rhesus monkeys. IV: Responses to a novel environment and to an alien species. J. comp. physiol. Psychol. 55: 363-368, 1962. 10. Menzel, E. W., Jr., R. K. Davenport, Jr. and C. M. Rogers. The effects of environmental restriction upon the chimpanzee's responsiveness to objects. J. comp. physiol. Psychol. 56: 78-85, 1963. 11. Menzel, E. W., Jr., R. K. Davenport, Jr. and C. M. Rogers. Effects of environmental restriction upon the chimpanzee's responsiveness in novel situations. J. comp. physiol. Psychol. 56: 329-334, 1963. 12. Messer, M., E. B. Thoman, A. G. Terrasa and P. R. Dallman. Artificial feeding of infant rats by continuous gastric infusion. J. Nutri. 98: 404-410, 1969. 13. Miller, S. A. and D. M. Czajka. The influence of dietary osmolarity on survival in the neonatal rat. Biol. Neonate 11: 197-203, 1967. 14. Salas, M., C. Guzman-Flores and S. Schapiro. An ontogenetic study of olfactory bulb electrical activity in the rat. Physiol. Behav. 4: 699-703, 1969. 15. Schapiro, S. and M. Salas. Behavioral response of infant rats to maternal odor. Physiol. Behav. 5: 815-817, 1970.
Maternal and Nutritional Factors in Maintenance of Infant Rat Cardiac Rate Following Maternal Separatioff MANFERD D. KOCH 2 AND WILLIAM J. ARNOLD
Department o f Psychology, University o f Nebraska-Lincoln, Lincoln, ArE 68508 (Received 20 March 1975) KOCH, M. D. AND W. J. ARNOLD. Maternal and nutritional factors in maintenance of infant rat cardiac rate following maternal separation. PHYSIOL. BEHAV. 16(5) 521-527, 1976. - Nutrient intake frequency and the presence of a surrogate mother were studied as factors influencing preweanling rat heart rates following maternal separation. Three groups of pups were separated from lactating females at 4, 7, and 10 days of age and maintained for 16 hr under the conditions of high frequency nutrient intake, low frequency nutrient intake, sham fed with no nutrient intake, or both high frequency intake and presence of a foster mother. A group of 14-day-olds were tested under the first three nutrient intake conditions. Results indicated that both nutritional factors and presence and behavior of the mother are influential in maintaininghigh sympathetic tone in preweanling rats. Maternal separation
Heart rate
Sympathetic
Parasympathetic
Frequency of nutrient intake
normal basal heart rate levels. Through the process of disproving alternative hypotheses, Hofer and Weiner conclude that the aspect of deprivation which is responsible for the decelatory heart rate effect may lie within the feeding interaction. However, the possibility still exists that some combination for proper nutrient intake and presence of adequate maternal stimuli are necessary for optimal heart rate maintenance. The initial purpose of the present experiment was to determine whether frequency of nutrient intake is a crucial factor for maintaining high sympathetic tone and cardiac rate prior to weaning. Mother rats repeat a cycle of nursing and moving away from the pups throughout the rearing period prior to weaning [ 1]. Between 12 and 19 days of age the median nursing phase duration is 26 min and the mother is away from the pups for 54 min on the average [5]. Therefore, the periods between nutrient intake are of about 1 hr duration. In the present study it was hypothesized that frequencies of nutrient intake which approximate those occurring naturally, i.e., 1 per hr, would be effective in maintaining high resting heart rate levels, and that relatively low frequencies of nutrient intake which were used by Hofer [6], i.e., 1 feeding every 4 b_r, would result in decreasing heart rates following maternal separation although the same volume of formula was intubated over a 16 hr period. The second purpose of this experiment was to determine whether stimuli provided by the physical presence of a surrogate mother would be effective in elevating inactive heart rates under conditions where actual
STUDIES involving maternal deprivation have shown that depriving an infant of contact with its biological mother or adequate maternal substitute has deleterious effects on the offspring's ability to cope with stressful situations later in life [2, 8, 9, 10, 11]. Research is now beginning to show how specific physiological systems, such as the autonomic nervous system, may be directly altered by early maternal separation. Hofer [3] demonstrated that separating 14day-old rat pups from their natural mothers caused a decrease in the pups' active and inactive heart rates by 40% during the first 12 to 16 hr following separation. Although nutritional factors may be important, merely providing adequate nutrient intake to produce weight gains did not reverse bradycardia following maternal separation in this study. Hofer [4] reported a subsequent study which showed that reversal of the decrease in cardiac rate following 16 hr of maternal separation in 15-day-old rat pups is a function of the amount of milk intubated. The implication of this study is that cardiac rate appears to be controlled, at least in part, by quantitative and scheduling characteristics of nutrient intake. Hofer and Weiner [7] found no significant differences in heart rate functioning between pups housed with nonnursing mothers and those completely deprived of maternal contact. They conclude that the physiological changes resulting from maternal deprivation are not dependent on loss of the physical presence of the mother per se. These findings initially appear to diminish the likelihood that olfactory, auditory and tactile stimuli are essential for the maintenance of
aThis research was supported by Research Grants MH-17377-03 and MH-17377-04 from the National Institute of Mental Health, United States Public Health Service. 2Requests for reprints may be sent to Manferd D. Koch, Department of Psychology, University of Nebraska-Lincoln, Lincoln NE 68508. 521
522
KOCH AND ARNOLD
nursing was precluded but pups were provided with normal occurring frequencies of nutrient intake. A total of three age levels, 4-, 7-, and 10-day-olds, were studied under each of the following conditions: (a) High Frequency (HF) group - animals tube fed bovine formula every hour following separation from their natural mothers; (b) Low Frequency (LF) group - animals tube fed once every 4th hour following separation and sham fed once every other hour; (c) Sham Fed (SF) group - animals sham fed only once very hour following sepataion; and (d) Foster Mother (FM) group - animals tube fed every hour following separation and housed with a foster mother whose teats had been surgically excised to preclude nursing. An additional group of 14-day-old pups was tested in an attempt to replicate in part Hofer's 1970 study in terms of the influence of nutritional factors on heart rate at that age. METHOD Animals and Surgery Twenty-five time-pregnant random-bred females with known dates of sperm positivity were obtained from the Charles River Company. During the experiment the females were housed in individual cages on ad lib food and water. Of these females, 24 delivered within 12 hr. Twenty-four hr prior to separating the pups from their mothers for testing, each pup was removed from the nest and two electrodes were implanted subcutaneously under light ether anesthesia. The electrodes consisted of fine 36 ga stainless steel wires which were implanted and formed into small loops with twisted ends. One electrode was positioned dorsally at right angles to the spine posterior to the forelegs and the second dorsally at right angles to the spine directly over the hind legs. Collodion was applied around the skin areas where the electrodes emerged to prevent recording artifacts due to electrode movement. The electrode implanting procedure took approximately 3 min; immediately after which the pups were returned to their home nests. Starting with litters born first, 40 pups were drawn from the total pool of male pups in the first 8 litters and were randomly assigned to the four experimental conditions at 4 days of age. The procedure of assigning pups to age levels on the basis of birth order was continued for age levels 7 and 10. The HF, LF, SF, and FM groups sizes for the three age levels were as follows: 4 days, n = 10; 7 days, n = 1 0 ; a n d 10 days, n = 11. This resulted in 124 pups being used. Animals were not disturbed between parturition and testing except for electrode implantation. A female which had previously reared two litters, the last of which was weaned 60 days prior to this study, was used as the foster mother. Thirty days prior to this experiment the female was placed under ether anesthesia and her teats were surgically excised to preclude nursing. Apparatus and Procedure When the pups reached their respective test ages, they were removed from their home cages and mothers, weighed, and marked for identification purposes. Since pups younger than 16 days are not homeothermic and body temperature is known to affect the cardiac intrinsic pacemaker [6], pups in the HF, LF, and SF groups were placed in incubators (20 x 26 x 15 cm) which were kept at a constant temperature between 32°C and 33°C. Maintenance of this temperature has been found to be of the
greatest importance for successful rearing of rat pups in the absence of a nesting female [12]. The temperature of the pups in the FM groups was maintained by the body heat from the foster mother; she crouched over the pups for long periods of time as if she were nursing them. Room temperature was maintained between 25°C and 27°C, and humidity was kept at 45%. The HF, LF and FM groups were fed a bovine milk formula consisting of 200 ml condensed milk, 200 ml distilled water, 40 g of instant dry milk, 2 cc Vi-Syneral, and the contents of one capsule of commercially-produced Methischol. The milk formula differs from that previously used by the investigators [8] in both water and instant dry milk content. The composition of the formula and volumes intubated at each feeding were based upon amounts which have previously been found sufficient to produce weight gains in long-term separated rat pups. Volumes of formula fed the three experimental groups at the three age levels are given in Table 1. (These volumes were prorated so that the groups of equal age received equal amounts of nutrient during the 16 hr of separation.) Pups of the FM group were fed the milk formula at rates and volumes identical to the HF group pups at the same age level. A stomach catheter, made of Silastic medical grade tubing (0.012 in I.D. and 0.025 in O.D.), reinforced with an inserted stainless steel suture wire, was used to intubate the formula at each feeding. The feeding process used involved inserting a feeding tube through the mouth into the stomach and injecting the appropriate volumes of formula via a syringe. The shamfeeding process used in both the SF and LF groups involved inserting a feeding tube through the mouth into the stomach of the pup for a period equal to that used for actual tube feeding in the HF, LF, and FM groups; however, no milk was injected. The sham-feeding process served as a control to equate handling and oral stimulation across the treatment groups. TABLE 1 VOLUMES OF BOVINE FORMULA FED ANIMALS IN EACH OF THREE EXPERIMENTALGROUPSAT FOUR AGE LEVELS Age in Days
HF
Experimental groups LF
FM
4 7 10 14
0.15 cc/hr 0.20 cc/hr 0.30 cc/hr 0.40 cc/hr
0.60 cc/4 hr 0.80 cc/4 hr 1.20 cc/4 hr 1.60 cc/4 hr
0.15 cc/hr 0.20 cc/hr 0.30 cc/hr --
Heart-rate recordings were taken at 0, 8, and 16 hr following separation from the mother using a Beckman Dynograph Recorder (Type RS) and a cardiotachometer coupler (Type 9857B). Lightweight recording clips were attached to the twisted ends of the implanted electrodes with a minimum amount of disturbance to the pup. The foster mother was gently removed from the nest just prior to each recording session and returned immediately after all measures were taken on FM group pups. Any movement on the part of the pup being recorded was noted on the chart paper and heart rates during periods of activity were not used. The pups were allowed approximately 30 sec to adapt to the placement of the recording clips prior to any recording. If a pup did not adapt, the clips were removed
MATERNAL SEPARATION AND HEART RATE
523 TABLE 2
MEAN BODY WEIGHTSAT SEPARATIONFROM THE MOTHERSAND AFTER 16 HR OF SEPARATION FOR ALL GROUPSSTUDIEDAT FOUR AGE LEVELS
Age in days 4 7 10 14
Mean Weights in Grams (S.D.) LF group SF group 0 hr 16 hr 0 hr 16 hr
HF group 0 hr 16 hr 11.9(1.0) 16.2(1.3) 22.3(2.1) 33.8(3.2)
14.2(1.1) 18.0(1.3) 24.9(2.0) 36.7(3.1)
11.9(1.1) 16.4(1.3) 21.9(2.0) 34.0(3.3)
14.0(1.1) 18.6(1.3) 24.5(1.9) 37.0(3.1)
and recordings were taken after the other pups had been recorded. Inactive heart rates during continuous 30 sec periods of recording constituted the measure of the dependent variable for subjects under all conditions. Recordings were always taken prior to the next feeding. RESULTS
Four-Day-OM Pups Mean body weights of 4-day-old pups when separation from the mother and after 16 hr of separation are given in Table 2. No significant difference among pup weights at the time of separation was found using a one way analysis of variance. However, a one way analysis of covariance of pups' weights after separation, using weights at the time of separation as the covariate, indicated a highly significant difference among groups, F(3,35) = 341.86, p<0.001. The
12.0(1.1) 16.3(1.4) 21.1(2.3) 33.4(2.8)
FM group 0 hr 16 hr
11.3(1.0) 11.7(0.9) 12.5(0.8) 15.6(1.3) 16.2(1.6) 17.3(1.4) 20.1(2.2) 22.6(2.0) 23.8(1.8) 31.4(2.6) ---
SF animals lost weight over the 16 hr of separation, and the groups which were fed gained weight. Animals housed with a foster mother did not gain as much weight as those in the other two groups, although they were fed identical quantities of milk. The lower weight gains of the FM group may be the result of greater activity induced by the presence and behavior of the foster mother, greater elimination due to the mother stimulating the anogenital area, altered autonomic nervous system functioning, or a combination of these three factors. Mean resting heart rates for the four groups at 0, 8, and 16 hr following separation from the mother are given in Fig. 1. A one way analysis of variance of group heart rates at 0 hr only approached significance, F(3,39) = 2.11, p>0.05. Because these initial heart rate means among groups were not equal, a 4 x 2 analysis of covariance was used to test for significance of group differences at 8 and 16 hr following separation. The overall
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380'
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340' Z
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H F GROUP " L F GROUP o-. . . . .
.-o
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,4
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--
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HOURS AFTER SEPARATION
FIG. 1. Cardiac responses of 4-day-old rat pups following separation from their natural mothers.
524 analysis of covariance on resting heart rates, using rates at 0 hr as the covariant, indicated that the differences among the groups were highly significant, F(3,36) = 17.79, p<0.005. A 4 × 2 analysis of variance indicated that the Hours and Groups × Hours factors were not significant. Orthogonal comparisons of covariance adjusted group heart rates over 8 and 16 hr indicated that the LF group had significantly higher mean rates than the HF group, F(1,36) = 9.11, p<0.005. Additional orthogonal comparisons indicated that the mean heart rates of the FM groups were significantly higher than the average of the two other groups which were fed (HF and LF), F(1,36) = 12.09, p<0.005. The average mean heart rates for the HF, LF, and FM groups was significantly higher than the mean heart rate of the SF group, F(1,36) = 25.90, p<0.005. The results did not support the hypothesis that normal occurring frequency of feeding was an important variable in maintenance of high sympathetic tone. However, contrary to results reported by Hofer [3], whether or not the pups were fed following separation did influence cardiac rate. The nutrient factor appears to be effected in maintaining sympathetic activity above that for pups totally deprived of food under conditions of long-term separation. AdditionaUy, the results indicated that stimuli associated with the presence of a maternal surrogate were sufficient to accelerate heart rates above levels occurring in that group shortly after separation from the natural mother and to increase heart rates significantly above those merely fed. Stimuli associated with the physical presence and behavior of the mother appear to be essential for the maintenance of high sympathetic tone. The foster mother may have provided more stimulation in attempting to nurse the pups than would have occurred under normal conditions. Because her nursing behavior was not regulated by milk release and uptake, she may have actually spent more time attempting to nurse than a lactating and nursing mother. Other research suggests that the total amount of time nonnutritive mothers spend with their pups may be higher than normal and that the rhythmic o n - o f f nursing pattern may be disrupted [7]. The possibility exists that in the FM condition an interaction effect between nutrient intake and stimuli of the surrogate mother accounts for a portion of the increases sympathetic tone and lower weight gains. Maternal stimuli, in the presence of nutriment, may have initially resulted in an increase in sympathetic discharge, achiorhydria and resultant decreased gastric and intestinal motility. The subsequent hourly intubations of nutriment, under conditions of diminished digestion, may have produced a greater arousal effect resulting in additionally increased sympathetic activity and tachycardia. The consequence of diminished digestion would be lower weight gains, as was observed in the FM pups.
Seven-Day-Old Pups Mean body weights among the four experimental groups over the 16 hr of separation showed the same general trends as were found for 4-day-old pups under similar rearing conditions (see Table 2). Again, no significant differences were found among group pup weights at 0 hr. Mean changes in body weights showed that FM pups gained less weight than HF or LF pups and that SF pups lost weight during the 16 hr period of separation. A one way analysis of covariance, using weights at the time of separation as the covariate, indicated a highly significant difference among
KOCH AND ARNOLD these group weights, F(3,35) = 260.44, p<0.001. Heart rate patterns similar to those of 4-day-olds were found for 7-day-old rat pups during the period of separation (see Fig. 2). Group heart rates at 0 hr did not differ significantly, but a 4 x 2 analysis of covariance of heart rates at 8 and 16 hr indicated that differences among the four groups were significant, F(3,36) --- 48.79, p<0.005. A 4 x 2 analysis of variance indicated that the Hours factor was not significant, although a significant Group x Hours factor was found, F(3,35) = 5.08, p<0.01, reflecting the rate increase in the HF and LF groups and the rate decrease in the SF group between 8 and 16 hr. Orthogonal comparisons of covariance adjusted group heart rates indicated that the difference between the HF and LF groups was not significant. Heart rate means of the FM group were significantly higher than the average of the HF and LF group means, F(1,36) = 38.42, p<0.005, again indicating that presence of maternal surrogate factors increases heart rates above those which occur when pups are simply provided adequate nutrient. The average of mean heart rates for all groups which were fed was significantly greater than the mean of the SF group, F(1,36) = 64.32, p<0.005, again implicating nutritional factors as important in maintaining heart rate.
Ten-Day-Old Pups The results of testing 10-day-old pups generally confirmed the results of 4- and 7-day-old pups reared under similar conditions. No significant differences existed among group pup weights at 0 hr. FM subjects gained less weight than HF or LF animals and SF animals lost weight during the period of separation (see Table 2). A one way analysis of covariance, using weights at the time of separation as the covariate, indicated a highly significant difference among the group weights after 16 hr of separation, F(3,39) = 530.18, p<0.001. Heart rate patterns, similar to those previously described for the other age levels studied, were found for 1-day-old pups (see Fig. 3). Group heart rates at 0 hr did not differ significantly, but a 4 x 2 analysis of covariance on resting heart rates at 8 and 16 hr indicated that the differences among the four groups were significant, F(,3,40) = 48.83, p<0.005. A 4 × 2 analysis of variance showed that the Groups x Hours interaction was significant, F(3,39) = 12.83, p<0.005, reflecting a decrease over hours for all groups except the FM group which increased. The Hours factor was not significant. Orghogonal comparisons of covariance adjusted group heart rates indicated a nonsignificant difference between the HF and LF groups, a significant difference between the combined HF and LF groups as contrasted to the FM groups, F(1,40) = 51.50, p<0.005, and a significant difference between the average of heart rate means of the fed groups contrasted to the heart rate mean of the SF group, F ( 1 , 4 0 ) = 51.35, p<0.005.
Fourteen-Day-Old Pups Hofer [3] reported that under the experimental conditions he used, physiological changes of 14-day-old pups following separation from the mother were not dependent upon starvation and dehydration. Because results of the current study indicated that nutrient intake was effective in maintaining cardiac rates at the 4-, 7-, and 10-day levels, a second segment of this study was conducted to determine if the nutritional factor was important at the 14-day-old level
MATERNAL SEPARATION AND HEART RATE
525
400-
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~g
H F GROUe :
280-
~'~'m
:
I. F GROUP o - . . . . o S F GROUP mm-. . . . . -m F M GROUP ±
1
260-
6
16
16.5
HOURS AFTER SEPARATION
FIG. 2. Cardiac responses of 7-day-old rat pups following separation from their natural mothers.
400-
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~m
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320'
300.
280. GROUI~ o-. . . . - o S F GROUP m-. . . . . • F M GROUP ±
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FIG. 3. Cardiac responses of 10-day-old rat pups following separation from their natural mothers.
526
KOCH AND ARNOLD
400.
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~
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H F GROUP : L F GROUP o- . . . . . S F GROUP • . . . . . .
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8 HOURSAFTERSEPARATION
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FIG. 4. Cardiac responses of 14-day-old rat pups following separation from their natural mothers. under the experimental conditions employed in this study. Three groups were studied: (a) high frequency; (b) low frequency; and (c) sham fed. Volumes of formula intubated in the HF and LF groups are given in Table 1. Mean body weight at separation and after 16 hr are given in Table 2. A one way analysis of variance indicated no significant difference among pup weights at 0 hr. An analysis of covariance of weights at 16 hr, using weights at the time of separation as the covariate, indicated a highly significant difference among the group weights, F(2,23) = 117.18, p<0.001, although there was only a small difference between the mean weights of the groups which were fed. Mean heart rates for the three groups are given in Table 4. A one way analysis of variance of group heart rates at 0 hr only approached significance, F(2,24)-3.35, p>0.05. An analysis of covariance, using 0-hr heart rates as the covariate, indicated a significant overall difference at 8 and 16 hr, F(,2,24) = 8.65, p<0.005. The mean resting heart rates for the three groups decreased significantly over the 16 hr o f separation, F(1,23) = 23.47, p<0.005, although there was no significant Group × Hours interaction. The difference between the average of heart rate means for the HF and LF groups and the mean heart rate for the SF group was significant, F(1,24) = 12.52, p<0.005; the HF and LF did not differ significantly. These results are consistent with those of the other three age levels in showing that nutritional factors which prevent dehydration and starvation are important variables in the maintenance of cardiac rate in rat pups. DISCUSSION
Results of this study implicate at least two groups of
factors as critical in the maintenance of high sympathetic tone in rat pups prior to weaning: (a) nutritional factors, and (b) maternal stimuli. The first group of factors, nutritional, has at least a minimal influence on maintenance of heart rate levels. At each age level studied, heart rates for the groups artificially provided the milk formula were higher than for those merely sham fed. These results differ from those previously published by Hofer [3] in that he found no significant differences between the mean heart rates of 14-day-old pups which were fed every 4 hr and the heart rates of pups merely sham fed. Differences exist between the composition and volumes of milk formulae used in the two studies which could account for the different results. Hofer reportedly fed his pups nondiluted, reconstituted condensed milk which was considered to be sufficient to prevent starvation and dehydration, and it did produce weight gains. However, since condensed milk is slightly higher in dietary osmolarity (approximately 986 mosmols/1) than the formula used in the present study (approximately 901 mosmols/1), the former may have been less effective in maintaining heart rates because it was more dehydrating. Dehydration has been shown to reduce survival of neonatal rats [13]. A more marked difference which may explain the discrepant results between the earlier study and this research is the volume of formula fed during the period of separation. Hofer [ 3 ] fed each pup 0.8 ml of formula every 4 hr while in the present study twice that volume was fed - 1.6 cc/4 hr. The larger volumes may have been more effective in stimulating sympathetic activity since larger volumes ( 1 . 0 - 1 . 5 ml) have been shown to be more effective than smaller volumes (0.5 ml) in stimulating tachycardia in 14-day-old rat pups separated
MATERNAL SEPARATION AND H E A R T RATE
527
from their mothers for 16 hr prior to intubation [4]. The question of whether frequency of nutrient intake influences cardiac maintenance remains unanswered. Under the artificial feeding conditions used in this study, there appears to be little reliable difference between intubating formula once every 4 hr or once every hour. The only age level at which a significant difference occurred between the HF and LF condition was at 4 days of age, and that difference was in the direction opposite to that predicted, but no explanation is readily available for this unexpected difference. Hofer and Weiner [7] found that the mere presence of a nonlactating mother failed to prevent a decrease in heart rates over a 24 hr period. However, no attempt was made by the researchers to provide the pups with nutriment during the experiment. Hofer and Weiner hypothesized that it is possibly some aspect o f the feeding interaction with the mother that regulates sympathetic tone at high levels prior to weaning. Results of the present study suggest that high sympathetic tone is dependent on a combination of at least two factors, nutritional and maternal stimuli, but not necessarily nursing per se. The second factor, maternal stimuli, involves a complex of stimuli associated with the physical presence and behavior of the mother (or adequate maternal surrogate). Research has shown that heart rates occurring immediately following separation are somewhat depressed as compared with preseparation levels [6]. However in the present study when both factors, surrogate maternal stimuli and nutriment, were present, heart rates of pups at 4, 7, and 10 days of age appeared to accelerate toward what presumably were preseparation levels. The individual presence o f one of these two factors does not appear to be a sufficient stimulus to maintain sympathetic tone. These results suggest that in addition to the role of nutrition, maternal stimuli, such as stimulation of mic-
turation and defecation, physical contact, and possibly pheromones may be individually or in combination important in the regulation of autonomic functioning. It has been shown that rat pups as young as 2 days of age are able to behaviorally respond to maternal odors [ 14,15 ] which suggests the possibility that the FM pups in the present study may have been responding to olfactory cues associated with the mature female. Another possibility is that rat pups become conditioned to a total complex of stimuli associated with the adult female, and that these stimuli are capable of eliciting high sympathetic activity. One implication o f the findings of this study is that when the mother leaves the nest and is absent for a long period of time, which she must be in order to hunt for food under natural conditions, some physiological processes of the pups reverse. The result is that parasympathetic activity predominates causing a resultant lowering o f heart rate. This would be adaptive in decreasing activity and conserving energy. The longer the separation, the more marked the parasympathetic activity and the greater the resultant energy conservation. When the female returns to the nest and again initiates nursing, sympathetic activity would begin to predominate. A system of reciprocal, environmentally elicited autonomic responses would have high survival value for the species because high sympathetic activity should facilitate behaviors such as nipple-seeking and nursing, while parasympathetic activity would reduce random movement away from the safety of the nest. Another implication of these findings is the possibility that separating rat pups from their mothers for several days would affect the development of adult autonomic functioning. This is substantiated in part by Koch and Arnold's [8] finding that maternally deprived rats, as adults, showed aberrant autonomic reactivity to environmental stimuli as contrasted to rats reared with mothers.
REFERENCES 1. Grota, L. J. and R. Ader. Continuous recording of maternal behavior in Rattus Norvegicus. Anirrt Behav. 17" 722-729, 1969. 2. Harlow, H. F. and M. K. Harlow. Social deprivation in monkeys. Scient. Am. 207: 136-146, 1962. 3. Hofer, M. A. Physiological responses of infant rats to separation from their mothers. Science 168: 871-873, 1970. 4. Hofer, M. A. Cardiac rate regulated by nutritional factors in young rats. Science 172: 1039-1041, 1971. 5. Hofer, M. A. and M. Grabie. Cardiorespiratory regulation and activity patterns of rat pups studied with their mothers during the nursing cycle. Devel. Psychobiol. 4: 169-180, 1971. 6. Hofer, M. A. and M. F. Reiser. The development of cardiac rate regulation in preweanling rats. Psychosonz Med. 31: 372-388, 1969. 7. Hofer, M. A. and H. Weiner. Development and mechanisms of cardio-respiratory responses to maternal deprivation in rat pups. Psychosom. Med. 33: 353-362, 1971. 8. Koch, M. D. and W. J. Arnold. Effects of early social deprivation on emotionality in rats. J. cornp, physiol. Psychol. 78: 391-399, 1972.
9. Mason, W. A. and P. C. Green. The effects of social restriction on the behavior of rhesus monkeys. IV: Responses to a novel environment and to an alien species. J. comp. physiol. Psychol. 55: 363-368, 1962. 10. Menzel, E. W., Jr., R. K. Davenport, Jr. and C. M. Rogers. The effects of environmental restriction upon the chimpanzee's responsiveness to objects. J. comp. physiol. Psychol. 56: 78-85, 1963. 11. Menzel, E. W., Jr., R. K. Davenport, Jr. and C. M. Rogers. Effects of environmental restriction upon the chimpanzee's responsiveness in novel situations. J. comp. physiol. Psychol. 56: 329-334, 1963. 12. Messer, M., E. B. Thoman, A. G. Terrasa and P. R. Dallman. Artificial feeding of infant rats by continuous gastric infusion. J. Nutri. 98: 404-410, 1969. 13. Miller, S. A. and D. M. Czajka. The influence of dietary osmolarity on survival in the neonatal rat. Biol. Neonate 11: 197-203, 1967. 14. Salas, M., C. Guzman-Flores and S. Schapiro. An ontogenetic study of olfactory bulb electrical activity in the rat. Physiol. Behav. 4: 699-703, 1969. 15. Schapiro, S. and M. Salas. Behavioral response of infant rats to maternal odor. Physiol. Behav. 5: 815-817, 1970.