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Acute Stress in Pregnant Rats
Physiology & Behavior, Vol. 62, No. 5, pp. 1087–1092, 1997 © 1997 Elsevier Science Inc. All rights reserved. Printed in the U.S.A. 0031-9384/97 $17.00 1 .00
PII S0031-9384(97)00261-8
Acute Stress in Pregnant Rats: Effects on Growth Rate, Learning, and Memory Capabilities of the Offspring B. LORDI,* P. PROTAIS,† D. MELLIER‡ AND J. CASTON1* *Laboratoire de Neurophysiologie Sensorielle, Faculte´ des Sciences, Universite´ de Rouen, 76821 Mont-Saint-Aignan Cedex, France, †Laboratoire de Physiologie, UFR de Me´decine et Pharmacie, Universite´ de Rouen, B.P. 97, 76803 Saint-Etienne du Rouvray Cedex, France, and ‡Laboratoire de Psychologie du De´veloppement, UFR de Psychologie-Sociologie, Universite´ de Rouen, 76821 Mont-Saint-Aignan Cedex, France Received 3 March 1997; Accepted 8 May 1997 LORDI, B., P. PROTAIS, D. MELLIER, AND J. CASTON. Acute stress in pregnant rats: Effects on growth rate, learning, and memory capabilities of the offspring. PHYSIOL BEHAV 62(5) 1087–1092, 1997.—Growth rate of the offspring of female rats stressed by the presence of a cat at the 10th or the 19th gestational day was lower than that of controls whereas footshocks administered at the same periods did not significantly influence growth rate of the young. Whatever the nature of the stress and the time when it was administered to the mother, the death rate of the young rats was much greater than that in controls. When adult, the offspring of stressed mothers exhibited learning and memory impairments in a delayed alternation task as well as in passive avoidance conditioning. Alteration of these cognitive functions is interpreted in terms of subtle dysfunctions in the development of the nervous system through modifications of the hormonal components of the mothers, particularly eventual alterations of the nervous system biochemistry of the offspring. © 1997 Elsevier Science Inc. Stress
Growth
Learning
Memory
Cognitive functions
MANY studies have investigated the effects of various acute and chronic stresses during pregnancy on the physiological health of the fetuses and newborn. Daily handling, exposure to sound or illumination stresses, and heat and restraint stresses applied at various times after mating block pregnancy (10,16,25,34), reduce the number of litters or the litter size in a number of mammals (11,35,36), and produce a high mortality of neonates soon after birth and survivors are small (12,20). Many stresses (cold, heat, footshocks, social stresses), when applied during pregnancy, result in lowered birthweights of the offspring (5,8,15,20,26). In women, psychological stresses provoke abortion and increase the percentage of premature infants; moreover, the delivery is often hard, the infants have lower birthweights, and the rate of pregnancy abnormality and neonatal pathology is higher than in unstressed mothers (4,13,24,27). It has also been demonstrated that besides the physiological alterations observed in the offspring of stressed mothers, such as bradycardia, arterial hypotension, hypoxia (18,19), digestive and pulmonary troubles, and hyperactivity (6,7,29), emotional stresses during pregnancy (as opposed to physical stresses which affect the physical integrity of the animal) can elicit an increased emotionality in the offspring (32) and cognitive def-
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To whom requests for reprints should be addressed.
icits in the newborn (31) as well as neurological and behavioral abnormalities (30). Neurological and behavioral deficits in the offspring of stressed mothers can sustain alterations of learning capabilities. Surprisingly, few studies have investigated this point. It was previously demonstrated (33), in the rat, that the offspring of mothers subjected from Day 5 to Day 18 of gestation to two audiogenic seizures per day were significantly lower in water maze learning than controls. It has also been demonstrated (28) that the offspring of stressed pregnant rats were inferior to controls in discrimination learning and that the errors they made in learning were greater than in controls. In these studies, the stresses were given chronically and data about the effects of acute stresses are lacking. The aim of the present investigation was therefore to study systematically, in the rat, the influence of two stresses (footshocks and the presence of a cat) administered on a given day during pregnancy (either the 10th day, when the neural tube is being formed, or the 19th day, i.e., 3 days before delivery, when all the gross nervous structures are achieved) on litter characteristics, growth of the newborn, and learning capabilities of the offspring. One stress (footshocks) could act directly on the fetuses, and the other (the presence of a cat) could not.
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LORDI ET AL. MATERIAL AND METHODS
1. Animals The animals were DA/HAN strain rats (pigmented rats), born in the laboratory and housed in standard conditions (12 h of light–12 h of dark, 20 –22°C, food and water ad lib). 2. Experimental Protocol Sixteen primiparous rats, about 4 months old, were submitted to a stress either at the 10th gestational day (G10: n 5 8), when the neural tube was being formed, or at the 19th gestational day (G19: n 5 8), i.e., 2 or 3 days before delivery, once the maturation of the nervous structures was well advanced. Three females and two males were put together in a cage in the evening and the following day, in the morning, the vaginal smear was examined. The presence in the smear of both vaginal cells typical of the oestrus stage and spermatozoids indicated the beginning of the gestation (Day 1). For both groups, G10 and G19, the stress was either electric footshocks or the presence of a cat close to the rat. Footshocks consisted of two series of 15-min continuous electric shocks (20 V, 3 mA, 30 Hz) spaced by a 15-min interval. They were delivered in a box 38 3 26 3 19 cm equipped with a grid floor. Whatever the series, the animal could not escape the nociceptive stimulus. The cat stress was elicited by putting a cat and a pregnant rat together in a wooden box (54 3 44 3 18 cm) for two periods of 15 min spaced by a 15-min interval. The experimenter continuously looked at the animals to prevent the rat from being strongly aggressed by the cat. Contrary to the rats stressed by the electric shocks, the animals stressed by the presence of the cat could, to some extent, avoid a close contact with the cat; they also could escape by jumping over the walls of the box and, if so, they were immediately returned to the box with the cat. In both stress conditions (nociceptive stimuli and the presence of the cat), the rats exhibited strong motor and autonomic reactions: jumps, squeaks, pilo-erection, behavioral inhibition, or motor excitation. After the stress was administered, the rats were returned to their cages until delivery. The day of birth (Day 0) was determined by examining the cages twice daily. The young rats were kept with their mother until the age of 6 weeks. The groups of young animals born from mothers that were stressed at the 10th gestational day or at the 19th gestational day were called S10 and S19, respectively, and the rats born from mothers stressed by the electric shocks or by the presence of the cat were called SE and SC, respectively. The four groups of experimental rats, which consisted only of males, were therefore the following: S10E (10 rats from 5 litters), S10C (10 rats from 3 litters), S19E (10 rats from 4 litters), and S19C (10 rats from 4 litters). A control (C) group consisted of 20 rats born from 5 mothers that were not stressed. 3. Litter Characteristics and Growth of the Young Rats Several parameters were measured in all the experimental rats as well as in controls: duration of gestation, number of young animals per litter, number of young rats that died the day of birth or during the following days, sex ratio (number of males/number of the animals in the litter), and evolution of the weight from Day 1 to Day 60. Given that all the learning experiments (vide infra) were performed on males, only growth of the males was studied. Useful comparisons of the number of young per litter, sex ratio, and death of pups were made according to the x2 test. Comparisons of the evolution of weight was made according to ANOVA.
4. Learning Experiments When 3 months old, the rats were subjected to two experimental protocols: first, a delayed alternation task and, second, a passive avoidance conditioning. This order of testing was the same for all the experimental and control animals, the interval between the two tests being 3 weeks. 4.1. Delayed alternation. The two-trial delayed alternation test was conducted in a T-maze (stem: 30 3 6 cm; arms: 17 3 6 cm; height of the walls: 10 cm), one lateral arm of which (either the left or the right one) could be blocked by a barrier. The first trial was a forced trial in that the animal placed in the stem could only explore and enter one arm of the T-maze, the other arm being blocked by the barrier. In the second trial (choice trial), both arms were accessible and the rat could either choose the same arm or alternate. Each rat was subjected to only one forced trial followed by a choice trial per day. The blocked arm was changed every day, that is the right side on Day 1, the left side on Day 2, the right side again on Day 3, etc. The intertrial interval was 30 s on Days 1, 4, and 7, 3 min on Days 2, 5, and 8, and 1 h on Days 3, 6, and 9. Between the trials, the floor and the walls of the T-maze were cleaned with alcohol to mask the olfactory cues. For each time delay between the forced and the choice trials, the percentage of delayed alternation was calculated, a 50% value meaning a complete loss of memory of the arm visited during the forced trial, the animals going to visit the left arm or the right arm at random. A 3 3 3 (groups 3 retention interval) ANOVA was used to test the eventual interaction in these data. Tests of single group measures against 50% (chance baseline) were made by matched-pair t-tests. 4.2. Passive avoidance conditioning. The experiments were conducted in a shuttle box separated into two compartments by an incomplete partition wall which allowed the animals to pass from one compartment to the other. One compartment (28 3 26 3 20 cm) was illuminated, the other (40 3 26 3 20 cm) being dark and equipped with a grid floor. A starting box (11 3 8 3 20 cm), joined to the lighted compartment, was separated from it by a sash-door. All the animals were isolated 24 h before the experiment to prevent a possible stress by sudden isolation when tested. A rat was placed in the starting box and the sash-door pulled up. After several seconds delay during which the animal explored the lighted compartment, it spontaneously entered the dark compartment. Once the rat’s four feet were in contact with the grid floor, the rat received electric shocks that lasted until it returned to the lighted compartment, a process that required 3– 4 s. The animal was allowed to stay in the lighted compartment for 1 min before returning to its cage in order for it to know that the dark compartment only was associated with a nociceptive stimulus. The time (T1) that elapsed from the moment when the rat entered the lighted compartment to the moment it entered the dark compartment was measured. After 7 days, the animals were subjected to a retrieval test given exactly in the same way as the initial conditioning, except for the fact that the rat did not get electric shocks when entering the dark compartment (these shocks were not necessary). The time (T2) from the moment the rat entered the lighted compartment to the moment it entered the dark compartment was measured. If the T2 2 T1 difference was zero or small, this indicated that at the time of the retrieval test, the animals had forgotten their initial nociceptive experience; conversely, if it was high, the animals had remembered their nociceptive experience. According to a previous study (9), we have assigned to T2 an upper value of T1 1 10 min. In other words, if the rat did not enter the dark compartment within T1 1 10 min when given the retrieval test, it was returned to its cage. For each group, the times T1 and T2 needed for each animal to
GROWTH AND COGNITION AFTER PRENATAL STRESS
1089 mental animals versus those of controls arbitrarily fixed to 100, one can observe that growth of S10E and S19E rats was roughly normal. In contrast, growth of S10C and S19C rats was slow. The following are noteworthy: (1) At Day 1, the mean weight of S10C rats was significantly lower (217%) than that of controls (F[1–28] 5 12.90, p , 0.01); at the same day, the weight of S19C rats was not (25.7% only: F[1–28] 5 2.53, NS). (2) The weight of S10C rats was lower than that of controls until the 50th day and reached control values by the 60th day only; in contrast, the weight of S19C rats, which was dramatically lower than that of controls until the 30th day (221.9% at Day 20 and 219.7% at Day 30), increased very quickly thereafter to reach control values by the 40th day. At Day 20, F[1–28] 5 19.89, p , 0.01; at Day 30, F[1–28] 5 8.09, p , 0.01. 2. Delayed Alternation
FIG. 1. Evolution of weights (ordinates) as a function of age, in days (abscissae), of the experimental groups relative to that of controls arbitrarily fixed to 100. Stars: values significantly different from those of controls; p , 0.05 (*), p , 0.01 (**), p , 0.001 (***).
The test was done in 60 animals (10 S10E, 10 S10C, 10 S19E, 10 S19C, and 20 C). For each given time interval between the forced and the choice trials, 3 measures were done (1 measure per day for 3 consecutive days); the percentage of spontaneous alternation was therefore calculated from 30 measures for each experimental group of 10 animals and from 60 measures for the control group of 20 animals (Fig. 2). In controls, the percentages of alternations were 83.3–91.7%, values that were significantly higher than that predicted by chance (Fig. 2 and Table 1A). The influence of the stress on the alternation behavior was similar whatever the nature of the stress (electrical
enter the dark compartment as well as the T2 2 T1 differences were respectively averaged, the mean values being given 6 SEM (s/ =n). Intergroup comparisons were made according to the Mann– Whitney test. RESULTS
1. Litter Characteristics and Growth Rate of the Young Rats The gestation time of the five control female rats was 21–22 days (mean 5 21.4 6 0.2 days). It was never shorter than 21 days and never longer than 22 days in the stressed female rats, and the mean values (21.1 6 0.1 days in G10 and 21.4 6 0.2 days in G19) were not significantly different from that calculated in controls. Although the number of young rats per litter was lower in stressed rats (4 –9 in G10: mean 5 7.0 6 0.7; 6 – 8 in G19: mean 5 6.7 6 0.4) than in controls (6 –10: mean 8.2 6 0.7), the differences were not significant. The sex ratio (number of males/total number of young rats per litter) was also lower in stressed rats (46.9% and 48.9% in G10 and G19 rats, respectively) than in controls (56.1%), but the differences were not significant. The death rate of the pups was higher in G10 (22.4%) and G19 (20.0%) than in controls (4.9%). The difference between G10 and controls is significant (x2 5 5.75, df 5 1, p , 0.05), as is the difference between G19 and controls (x2 5 4.59, df 5 1, p , 0.05). In G10 as well as in G19 rats, death occurred mainly during the first 2 days (in 91.7% and 81.8% of the cases, respectively). It was not due to infanticide by the mothers and was therefore probably the result of physiological changes in the young rats. Among each of the two stressed groups, all these parameters were similar whatever the nature of the stress (electric shocks or the presence of the cat). Great differences do exist between growth of the experimental and control rats, the differences depending more on the nature of the stress than on the developmental stage when it was administered. Indeed, from Fig. 1, which depicts weights of the experi-
FIG. 2. Delayed alternation experiment. Percentages of alternations (6SEM) as a function of the time delay between the forced and the choice trials. Stars: significant difference relative to chance; p , 0.05 (*), p , 0.01 (**), p , 0.001 (***). No star indicates that the difference relative to chance is not significant.
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LORDI ET AL. TABLE 1A.
STATISTICAL COMPARISONS (MATCHED-PAIR t-TESTS) OF THE PERCENTAGE OF ALTERNATIONS IN C, S10, AND S19 RATS VERSUS CHANCE BASELINE (50%)
C S10 S19
30 s
3 min
1h
12.42*** 5.09*** 2.78*
9.76*** 3.77*** 3.39**
7.33*** 2.20* 0.002,NS
TABLE 1B. STATISTICAL COMPARISONS (MATCHED-PAIR t-TESTS) OF THE PERCENTAGE OF ALTERNATIONS IN S10 AND S19 RATS VERSUS CONTROL RATS
S10 versus controls S19 versus controls
30 s
3 min
1h
2.06* 2.72*
2.92** 2.75*
3.20** 4.34***
FIG. 3. Passive avoidance conditioning: (A) mean values (6SEM) of T1 and T2; (B) percentage of rats in which T1 values were greater than 14 s. Stars: p , 0.05 (*), p , 0.01 (**). In (A), significant difference between T1 and T2; in (B), significant difference relative to controls.
The values indicated are those of t and the difference is significant at p , 0.05 (*), p , 0.01 (**), and p , 0.001 (***) or not significant (NS).
shocks or the presence of the cat). S10 animals alternated above chance at 30 s, 3 min, and 1 h (Fig. 2 and Table 1A) but the percentages of alternations were always significantly lower than in controls (Table 1B). S19 rats alternated above chance at 30 s and 3 min only and not at 1 h (Fig. 2 and Table 1A). For these three time intervals, they alternated significantly less than controls (Table 1B). There was no significant group 3 delay interaction (F[4 –171] 5 0.62, p 5 0.64, NS). 3. Passive Avoidance Conditioning 3.1. T1 values during the initial conditioning. The exploration durations of the lighted compartment during the initial conditioning (T1 values) in control rats were short; in most of the animals (16 out of 20), they ranged between 2 and 14 s. In the four others, they were much longer, the longest one being 165 s. The mean T1 values in S10E (42.5 6 14.8 s) and S10C rats (95.9 6 48.7 s) were higher than that calculated in controls (25.6 6 9.7 s). The difference between S10E and control rats was significant (U 5 2.17, p , 0.05), as was the difference between S10C and control rats (U 5 2.83, p , 0.01) (Fig. 3A). Moreover, the numbers of rats for which T1 values were greater than 14 s were higher in S10E (70%) and S10C (80%) than in controls (20%) (Fig. 3B). The difference between S10E and control rats was significant (x2 5 7.18, p , 0.01), as was the difference between S10C and control rats (x2 5 10.00, p , 0.01). T1 values in S19E (3–397 s: mean 5 96.2 6 42.9 s) and S19C rats (4 –712 s: mean 5 123.5 6 67.9 s) were higher than in controls (2–165 s: mean 5 25.6 6 9.7 s) (Fig. 3A), but the differences were not significant. However, the number of rats for which T1 values were greater than 14 s were higher in S19E (50%) and S19C (60%) than in controls (20%), the difference being significant between S19C and C rats only (x2 5 4.80, p , 0.05). These results obviously demonstrate that S10 and S19 rats spent much more time exploring the lighted compartment of the shuttle box during the initial conditioning than controls. 3.2. Retention of the initial conditioning. No control rat studied entered the dark compartment during the retrieval test. Consequently, their absolute memory score (T2 2 T1) was maximal (600 s). Except for the S19E group, in which no rat entered the dark
compartment during the retrieval test, some animals of the other experimental groups did enter the dark compartment (1/10 in S10E, 2/10 in S10C, and 3/10 in S19C). Consequently, in all three of these groups, the absolute memory score was not maximal (Fig. 4). This suggests that the retention that the experimental animals had on their nociceptive experience was altered, except for the S19E group. DISCUSSION
Chronic stresses during pregnancy may provoke abortion, reduce litter size in a variety of mammalian species (11,12,20,35,36), and induce cognitive abnormalities in the offspring (Lordi et al., in preparation). Our results confirm that acute stresses have such drastic effects; indeed, whether or not litter size and sex ratio were different from those of controls, the percentage of death in young rats was dramatically high (about 20%) compared to normal. Moreover, such acute stresses reduced birthweight of the offspring, especially when the stressful agent was the presence of the cat, showing that a purely emotional stress had greater effects than electrical footshocks. In this condition, an early stress had more drastic effects on birthweight than a late stress. It could be ex-
FIG. 4. Passive avoidance conditioning. Mean values, in seconds (6SEM), of the absolute memory scores (T2 2 T1).
GROWTH AND COGNITION AFTER PRENATAL STRESS pected that smaller litter size, due to abortion or to early death of the newborn, would have resulted in larger pups and in an enhanced growth. As this was obviously not the case, it is conceivable that physiological disorders following stress in the pregnant rat greatly altered fetal growth when they happened early during the gestational period and that they had only mild effects on fetal growth when they happened later because it was almost achieved before the stress experience. The fact that stress in pregnant animals induces low birthweights is well known (5,8,12,15,20,26). Hormonal factors are involved in fetal growth and a proper mother–fetus hormonal balance is needed to ensure normal intra-uterine growth. Stress alters the blood concentration of hormones released by the hypothalamo–adenohypophyso–adrenal (23),–thyroid (21), and– gonadal (22) axes in the rat. Moreover, growth retardation might also be due to fetal oxygen supply, modifications of which may be the result of vascular changes due to alterations in sympathetic activity (3). S10C rats hardly recovered a weight similar to that of controls, the neonatal handicap being overcome only when they were adult. In S19C rats, whose birthweight was not significantly different from that of controls, the postnatal weight increased much more slowly than that of S10C and controls until 1 month of age. After that, the animals rapidly recovered a normal weight. This is most likely due to the fact that stressed mothers did not take care of their offspring, as suggested by experiments in progress. Moreover, when the animals were able to eat by themselves (by 1 month of age), they rapidly recovered a normal weight. To distinguish the effects of stresses in utero versus postnatal effects of maternal care, cross-fostering is probably an appropriate method. However, this method is likely to introduce a bias since cross-fostered animals are probably stressed. Such experiments are in progress to try to quantify the stress introduced by cross-fostering.
1091 Following a stress of the mother, the animals needed a long time to explore their environment. Indeed, the number of animals that explored the lighted compartment of the shuttle box for a long time was significantly higher than that of controls. Such a low activity and a low level of exploration behavior were also noted previously (1,32). Moreover, spatial memory was altered on a one-trial learning experiment: in the delayed alternation task, the experimental animals, whatever the group, had forgotten the arm visited 1 h before and had partially forgotten the arm visited 3 min before. Finally, the passive avoidance conditioning test also demonstrated a partial alteration of memory in the experimental groups. These results suggest that an acute stress at Day 10 or 19 of gestation in the rat influences the development of cognitive functions of the offspring, but one can only speculate about such effects. It is possible that the stresses generated by electric footshocks may act on the fetuses directly, but this can hardly be the case for the cat stress, which is exclusively emotional in nature. It seems more likely that the alterations of the cognitive functions were due to delayed growth rate (at least in the offspring of mothers stressed by the cat) as well as to deficits in maternal care and/or to subtle alterations in the development of the nervous system through modifications of the hormonal components of the mothers. It has already been said that a proper mother–fetus hormonal balance is needed for the development of the fetuses and it has also been suggested that stress during pregnancy might be a predisposing factor to mental illness (14,17) and to increased depression-related behavior in the female offspring during adulthood (2). Experiments are intended to try to correlate cognitive defects of young rats with modifications of the mother–fetus hormonal balance and with eventual alterations of their nervous system biochemistry.
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LORDI ET AL. 30. Stott, D. H. Follow-up study from birth of the effects of prenatal stresses. Dev. Med. Child Neurol. 15:770 –787; 1973. 31. Stott, D. H.; Camb, M. A.; Lond, P. D. Physical and mental handicaps following a disturbed pregnancy. Lancet 18:1006 –1012; 1957. 32. Thompson, W. R. Influence of prenatal maternal anxiety on emotionality in young rats. Science 125:698 – 699; 1957. 33. Thompson, W. D., Jr.; Sontag, L. W. Behavioral effects in the offspring of rats subjected to audiogenic seizures during the gestational period. J. Comp. Physiol. Psychol. 49:454 – 456; 1956. 34. Weir, M. W.; De Fries, J. C. Blocking of pregnancy in mice as a function of stress. Psychol. Reprod. 13:365–366; 1963. 35. Wildt, D. E.; Riegle, G. D.; Dukelow, W. R. Physiological temperature response and embryonic mortality in stressed swine. Am. J. Physiol. 229:1471–1475; 1975. 36. Zondek, B.; Tamari, I. Effects of auditory stimuli on reproduction. Ciba Found. Study Group 26:4 –19; 1967.
PII S0031-9384(97)00261-8
Acute Stress in Pregnant Rats: Effects on Growth Rate, Learning, and Memory Capabilities of the Offspring B. LORDI,* P. PROTAIS,† D. MELLIER‡ AND J. CASTON1* *Laboratoire de Neurophysiologie Sensorielle, Faculte´ des Sciences, Universite´ de Rouen, 76821 Mont-Saint-Aignan Cedex, France, †Laboratoire de Physiologie, UFR de Me´decine et Pharmacie, Universite´ de Rouen, B.P. 97, 76803 Saint-Etienne du Rouvray Cedex, France, and ‡Laboratoire de Psychologie du De´veloppement, UFR de Psychologie-Sociologie, Universite´ de Rouen, 76821 Mont-Saint-Aignan Cedex, France Received 3 March 1997; Accepted 8 May 1997 LORDI, B., P. PROTAIS, D. MELLIER, AND J. CASTON. Acute stress in pregnant rats: Effects on growth rate, learning, and memory capabilities of the offspring. PHYSIOL BEHAV 62(5) 1087–1092, 1997.—Growth rate of the offspring of female rats stressed by the presence of a cat at the 10th or the 19th gestational day was lower than that of controls whereas footshocks administered at the same periods did not significantly influence growth rate of the young. Whatever the nature of the stress and the time when it was administered to the mother, the death rate of the young rats was much greater than that in controls. When adult, the offspring of stressed mothers exhibited learning and memory impairments in a delayed alternation task as well as in passive avoidance conditioning. Alteration of these cognitive functions is interpreted in terms of subtle dysfunctions in the development of the nervous system through modifications of the hormonal components of the mothers, particularly eventual alterations of the nervous system biochemistry of the offspring. © 1997 Elsevier Science Inc. Stress
Growth
Learning
Memory
Cognitive functions
MANY studies have investigated the effects of various acute and chronic stresses during pregnancy on the physiological health of the fetuses and newborn. Daily handling, exposure to sound or illumination stresses, and heat and restraint stresses applied at various times after mating block pregnancy (10,16,25,34), reduce the number of litters or the litter size in a number of mammals (11,35,36), and produce a high mortality of neonates soon after birth and survivors are small (12,20). Many stresses (cold, heat, footshocks, social stresses), when applied during pregnancy, result in lowered birthweights of the offspring (5,8,15,20,26). In women, psychological stresses provoke abortion and increase the percentage of premature infants; moreover, the delivery is often hard, the infants have lower birthweights, and the rate of pregnancy abnormality and neonatal pathology is higher than in unstressed mothers (4,13,24,27). It has also been demonstrated that besides the physiological alterations observed in the offspring of stressed mothers, such as bradycardia, arterial hypotension, hypoxia (18,19), digestive and pulmonary troubles, and hyperactivity (6,7,29), emotional stresses during pregnancy (as opposed to physical stresses which affect the physical integrity of the animal) can elicit an increased emotionality in the offspring (32) and cognitive def-
1
Rat
To whom requests for reprints should be addressed.
icits in the newborn (31) as well as neurological and behavioral abnormalities (30). Neurological and behavioral deficits in the offspring of stressed mothers can sustain alterations of learning capabilities. Surprisingly, few studies have investigated this point. It was previously demonstrated (33), in the rat, that the offspring of mothers subjected from Day 5 to Day 18 of gestation to two audiogenic seizures per day were significantly lower in water maze learning than controls. It has also been demonstrated (28) that the offspring of stressed pregnant rats were inferior to controls in discrimination learning and that the errors they made in learning were greater than in controls. In these studies, the stresses were given chronically and data about the effects of acute stresses are lacking. The aim of the present investigation was therefore to study systematically, in the rat, the influence of two stresses (footshocks and the presence of a cat) administered on a given day during pregnancy (either the 10th day, when the neural tube is being formed, or the 19th day, i.e., 3 days before delivery, when all the gross nervous structures are achieved) on litter characteristics, growth of the newborn, and learning capabilities of the offspring. One stress (footshocks) could act directly on the fetuses, and the other (the presence of a cat) could not.
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1088
LORDI ET AL. MATERIAL AND METHODS
1. Animals The animals were DA/HAN strain rats (pigmented rats), born in the laboratory and housed in standard conditions (12 h of light–12 h of dark, 20 –22°C, food and water ad lib). 2. Experimental Protocol Sixteen primiparous rats, about 4 months old, were submitted to a stress either at the 10th gestational day (G10: n 5 8), when the neural tube was being formed, or at the 19th gestational day (G19: n 5 8), i.e., 2 or 3 days before delivery, once the maturation of the nervous structures was well advanced. Three females and two males were put together in a cage in the evening and the following day, in the morning, the vaginal smear was examined. The presence in the smear of both vaginal cells typical of the oestrus stage and spermatozoids indicated the beginning of the gestation (Day 1). For both groups, G10 and G19, the stress was either electric footshocks or the presence of a cat close to the rat. Footshocks consisted of two series of 15-min continuous electric shocks (20 V, 3 mA, 30 Hz) spaced by a 15-min interval. They were delivered in a box 38 3 26 3 19 cm equipped with a grid floor. Whatever the series, the animal could not escape the nociceptive stimulus. The cat stress was elicited by putting a cat and a pregnant rat together in a wooden box (54 3 44 3 18 cm) for two periods of 15 min spaced by a 15-min interval. The experimenter continuously looked at the animals to prevent the rat from being strongly aggressed by the cat. Contrary to the rats stressed by the electric shocks, the animals stressed by the presence of the cat could, to some extent, avoid a close contact with the cat; they also could escape by jumping over the walls of the box and, if so, they were immediately returned to the box with the cat. In both stress conditions (nociceptive stimuli and the presence of the cat), the rats exhibited strong motor and autonomic reactions: jumps, squeaks, pilo-erection, behavioral inhibition, or motor excitation. After the stress was administered, the rats were returned to their cages until delivery. The day of birth (Day 0) was determined by examining the cages twice daily. The young rats were kept with their mother until the age of 6 weeks. The groups of young animals born from mothers that were stressed at the 10th gestational day or at the 19th gestational day were called S10 and S19, respectively, and the rats born from mothers stressed by the electric shocks or by the presence of the cat were called SE and SC, respectively. The four groups of experimental rats, which consisted only of males, were therefore the following: S10E (10 rats from 5 litters), S10C (10 rats from 3 litters), S19E (10 rats from 4 litters), and S19C (10 rats from 4 litters). A control (C) group consisted of 20 rats born from 5 mothers that were not stressed. 3. Litter Characteristics and Growth of the Young Rats Several parameters were measured in all the experimental rats as well as in controls: duration of gestation, number of young animals per litter, number of young rats that died the day of birth or during the following days, sex ratio (number of males/number of the animals in the litter), and evolution of the weight from Day 1 to Day 60. Given that all the learning experiments (vide infra) were performed on males, only growth of the males was studied. Useful comparisons of the number of young per litter, sex ratio, and death of pups were made according to the x2 test. Comparisons of the evolution of weight was made according to ANOVA.
4. Learning Experiments When 3 months old, the rats were subjected to two experimental protocols: first, a delayed alternation task and, second, a passive avoidance conditioning. This order of testing was the same for all the experimental and control animals, the interval between the two tests being 3 weeks. 4.1. Delayed alternation. The two-trial delayed alternation test was conducted in a T-maze (stem: 30 3 6 cm; arms: 17 3 6 cm; height of the walls: 10 cm), one lateral arm of which (either the left or the right one) could be blocked by a barrier. The first trial was a forced trial in that the animal placed in the stem could only explore and enter one arm of the T-maze, the other arm being blocked by the barrier. In the second trial (choice trial), both arms were accessible and the rat could either choose the same arm or alternate. Each rat was subjected to only one forced trial followed by a choice trial per day. The blocked arm was changed every day, that is the right side on Day 1, the left side on Day 2, the right side again on Day 3, etc. The intertrial interval was 30 s on Days 1, 4, and 7, 3 min on Days 2, 5, and 8, and 1 h on Days 3, 6, and 9. Between the trials, the floor and the walls of the T-maze were cleaned with alcohol to mask the olfactory cues. For each time delay between the forced and the choice trials, the percentage of delayed alternation was calculated, a 50% value meaning a complete loss of memory of the arm visited during the forced trial, the animals going to visit the left arm or the right arm at random. A 3 3 3 (groups 3 retention interval) ANOVA was used to test the eventual interaction in these data. Tests of single group measures against 50% (chance baseline) were made by matched-pair t-tests. 4.2. Passive avoidance conditioning. The experiments were conducted in a shuttle box separated into two compartments by an incomplete partition wall which allowed the animals to pass from one compartment to the other. One compartment (28 3 26 3 20 cm) was illuminated, the other (40 3 26 3 20 cm) being dark and equipped with a grid floor. A starting box (11 3 8 3 20 cm), joined to the lighted compartment, was separated from it by a sash-door. All the animals were isolated 24 h before the experiment to prevent a possible stress by sudden isolation when tested. A rat was placed in the starting box and the sash-door pulled up. After several seconds delay during which the animal explored the lighted compartment, it spontaneously entered the dark compartment. Once the rat’s four feet were in contact with the grid floor, the rat received electric shocks that lasted until it returned to the lighted compartment, a process that required 3– 4 s. The animal was allowed to stay in the lighted compartment for 1 min before returning to its cage in order for it to know that the dark compartment only was associated with a nociceptive stimulus. The time (T1) that elapsed from the moment when the rat entered the lighted compartment to the moment it entered the dark compartment was measured. After 7 days, the animals were subjected to a retrieval test given exactly in the same way as the initial conditioning, except for the fact that the rat did not get electric shocks when entering the dark compartment (these shocks were not necessary). The time (T2) from the moment the rat entered the lighted compartment to the moment it entered the dark compartment was measured. If the T2 2 T1 difference was zero or small, this indicated that at the time of the retrieval test, the animals had forgotten their initial nociceptive experience; conversely, if it was high, the animals had remembered their nociceptive experience. According to a previous study (9), we have assigned to T2 an upper value of T1 1 10 min. In other words, if the rat did not enter the dark compartment within T1 1 10 min when given the retrieval test, it was returned to its cage. For each group, the times T1 and T2 needed for each animal to
GROWTH AND COGNITION AFTER PRENATAL STRESS
1089 mental animals versus those of controls arbitrarily fixed to 100, one can observe that growth of S10E and S19E rats was roughly normal. In contrast, growth of S10C and S19C rats was slow. The following are noteworthy: (1) At Day 1, the mean weight of S10C rats was significantly lower (217%) than that of controls (F[1–28] 5 12.90, p , 0.01); at the same day, the weight of S19C rats was not (25.7% only: F[1–28] 5 2.53, NS). (2) The weight of S10C rats was lower than that of controls until the 50th day and reached control values by the 60th day only; in contrast, the weight of S19C rats, which was dramatically lower than that of controls until the 30th day (221.9% at Day 20 and 219.7% at Day 30), increased very quickly thereafter to reach control values by the 40th day. At Day 20, F[1–28] 5 19.89, p , 0.01; at Day 30, F[1–28] 5 8.09, p , 0.01. 2. Delayed Alternation
FIG. 1. Evolution of weights (ordinates) as a function of age, in days (abscissae), of the experimental groups relative to that of controls arbitrarily fixed to 100. Stars: values significantly different from those of controls; p , 0.05 (*), p , 0.01 (**), p , 0.001 (***).
The test was done in 60 animals (10 S10E, 10 S10C, 10 S19E, 10 S19C, and 20 C). For each given time interval between the forced and the choice trials, 3 measures were done (1 measure per day for 3 consecutive days); the percentage of spontaneous alternation was therefore calculated from 30 measures for each experimental group of 10 animals and from 60 measures for the control group of 20 animals (Fig. 2). In controls, the percentages of alternations were 83.3–91.7%, values that were significantly higher than that predicted by chance (Fig. 2 and Table 1A). The influence of the stress on the alternation behavior was similar whatever the nature of the stress (electrical
enter the dark compartment as well as the T2 2 T1 differences were respectively averaged, the mean values being given 6 SEM (s/ =n). Intergroup comparisons were made according to the Mann– Whitney test. RESULTS
1. Litter Characteristics and Growth Rate of the Young Rats The gestation time of the five control female rats was 21–22 days (mean 5 21.4 6 0.2 days). It was never shorter than 21 days and never longer than 22 days in the stressed female rats, and the mean values (21.1 6 0.1 days in G10 and 21.4 6 0.2 days in G19) were not significantly different from that calculated in controls. Although the number of young rats per litter was lower in stressed rats (4 –9 in G10: mean 5 7.0 6 0.7; 6 – 8 in G19: mean 5 6.7 6 0.4) than in controls (6 –10: mean 8.2 6 0.7), the differences were not significant. The sex ratio (number of males/total number of young rats per litter) was also lower in stressed rats (46.9% and 48.9% in G10 and G19 rats, respectively) than in controls (56.1%), but the differences were not significant. The death rate of the pups was higher in G10 (22.4%) and G19 (20.0%) than in controls (4.9%). The difference between G10 and controls is significant (x2 5 5.75, df 5 1, p , 0.05), as is the difference between G19 and controls (x2 5 4.59, df 5 1, p , 0.05). In G10 as well as in G19 rats, death occurred mainly during the first 2 days (in 91.7% and 81.8% of the cases, respectively). It was not due to infanticide by the mothers and was therefore probably the result of physiological changes in the young rats. Among each of the two stressed groups, all these parameters were similar whatever the nature of the stress (electric shocks or the presence of the cat). Great differences do exist between growth of the experimental and control rats, the differences depending more on the nature of the stress than on the developmental stage when it was administered. Indeed, from Fig. 1, which depicts weights of the experi-
FIG. 2. Delayed alternation experiment. Percentages of alternations (6SEM) as a function of the time delay between the forced and the choice trials. Stars: significant difference relative to chance; p , 0.05 (*), p , 0.01 (**), p , 0.001 (***). No star indicates that the difference relative to chance is not significant.
1090
LORDI ET AL. TABLE 1A.
STATISTICAL COMPARISONS (MATCHED-PAIR t-TESTS) OF THE PERCENTAGE OF ALTERNATIONS IN C, S10, AND S19 RATS VERSUS CHANCE BASELINE (50%)
C S10 S19
30 s
3 min
1h
12.42*** 5.09*** 2.78*
9.76*** 3.77*** 3.39**
7.33*** 2.20* 0.002,NS
TABLE 1B. STATISTICAL COMPARISONS (MATCHED-PAIR t-TESTS) OF THE PERCENTAGE OF ALTERNATIONS IN S10 AND S19 RATS VERSUS CONTROL RATS
S10 versus controls S19 versus controls
30 s
3 min
1h
2.06* 2.72*
2.92** 2.75*
3.20** 4.34***
FIG. 3. Passive avoidance conditioning: (A) mean values (6SEM) of T1 and T2; (B) percentage of rats in which T1 values were greater than 14 s. Stars: p , 0.05 (*), p , 0.01 (**). In (A), significant difference between T1 and T2; in (B), significant difference relative to controls.
The values indicated are those of t and the difference is significant at p , 0.05 (*), p , 0.01 (**), and p , 0.001 (***) or not significant (NS).
shocks or the presence of the cat). S10 animals alternated above chance at 30 s, 3 min, and 1 h (Fig. 2 and Table 1A) but the percentages of alternations were always significantly lower than in controls (Table 1B). S19 rats alternated above chance at 30 s and 3 min only and not at 1 h (Fig. 2 and Table 1A). For these three time intervals, they alternated significantly less than controls (Table 1B). There was no significant group 3 delay interaction (F[4 –171] 5 0.62, p 5 0.64, NS). 3. Passive Avoidance Conditioning 3.1. T1 values during the initial conditioning. The exploration durations of the lighted compartment during the initial conditioning (T1 values) in control rats were short; in most of the animals (16 out of 20), they ranged between 2 and 14 s. In the four others, they were much longer, the longest one being 165 s. The mean T1 values in S10E (42.5 6 14.8 s) and S10C rats (95.9 6 48.7 s) were higher than that calculated in controls (25.6 6 9.7 s). The difference between S10E and control rats was significant (U 5 2.17, p , 0.05), as was the difference between S10C and control rats (U 5 2.83, p , 0.01) (Fig. 3A). Moreover, the numbers of rats for which T1 values were greater than 14 s were higher in S10E (70%) and S10C (80%) than in controls (20%) (Fig. 3B). The difference between S10E and control rats was significant (x2 5 7.18, p , 0.01), as was the difference between S10C and control rats (x2 5 10.00, p , 0.01). T1 values in S19E (3–397 s: mean 5 96.2 6 42.9 s) and S19C rats (4 –712 s: mean 5 123.5 6 67.9 s) were higher than in controls (2–165 s: mean 5 25.6 6 9.7 s) (Fig. 3A), but the differences were not significant. However, the number of rats for which T1 values were greater than 14 s were higher in S19E (50%) and S19C (60%) than in controls (20%), the difference being significant between S19C and C rats only (x2 5 4.80, p , 0.05). These results obviously demonstrate that S10 and S19 rats spent much more time exploring the lighted compartment of the shuttle box during the initial conditioning than controls. 3.2. Retention of the initial conditioning. No control rat studied entered the dark compartment during the retrieval test. Consequently, their absolute memory score (T2 2 T1) was maximal (600 s). Except for the S19E group, in which no rat entered the dark
compartment during the retrieval test, some animals of the other experimental groups did enter the dark compartment (1/10 in S10E, 2/10 in S10C, and 3/10 in S19C). Consequently, in all three of these groups, the absolute memory score was not maximal (Fig. 4). This suggests that the retention that the experimental animals had on their nociceptive experience was altered, except for the S19E group. DISCUSSION
Chronic stresses during pregnancy may provoke abortion, reduce litter size in a variety of mammalian species (11,12,20,35,36), and induce cognitive abnormalities in the offspring (Lordi et al., in preparation). Our results confirm that acute stresses have such drastic effects; indeed, whether or not litter size and sex ratio were different from those of controls, the percentage of death in young rats was dramatically high (about 20%) compared to normal. Moreover, such acute stresses reduced birthweight of the offspring, especially when the stressful agent was the presence of the cat, showing that a purely emotional stress had greater effects than electrical footshocks. In this condition, an early stress had more drastic effects on birthweight than a late stress. It could be ex-
FIG. 4. Passive avoidance conditioning. Mean values, in seconds (6SEM), of the absolute memory scores (T2 2 T1).
GROWTH AND COGNITION AFTER PRENATAL STRESS pected that smaller litter size, due to abortion or to early death of the newborn, would have resulted in larger pups and in an enhanced growth. As this was obviously not the case, it is conceivable that physiological disorders following stress in the pregnant rat greatly altered fetal growth when they happened early during the gestational period and that they had only mild effects on fetal growth when they happened later because it was almost achieved before the stress experience. The fact that stress in pregnant animals induces low birthweights is well known (5,8,12,15,20,26). Hormonal factors are involved in fetal growth and a proper mother–fetus hormonal balance is needed to ensure normal intra-uterine growth. Stress alters the blood concentration of hormones released by the hypothalamo–adenohypophyso–adrenal (23),–thyroid (21), and– gonadal (22) axes in the rat. Moreover, growth retardation might also be due to fetal oxygen supply, modifications of which may be the result of vascular changes due to alterations in sympathetic activity (3). S10C rats hardly recovered a weight similar to that of controls, the neonatal handicap being overcome only when they were adult. In S19C rats, whose birthweight was not significantly different from that of controls, the postnatal weight increased much more slowly than that of S10C and controls until 1 month of age. After that, the animals rapidly recovered a normal weight. This is most likely due to the fact that stressed mothers did not take care of their offspring, as suggested by experiments in progress. Moreover, when the animals were able to eat by themselves (by 1 month of age), they rapidly recovered a normal weight. To distinguish the effects of stresses in utero versus postnatal effects of maternal care, cross-fostering is probably an appropriate method. However, this method is likely to introduce a bias since cross-fostered animals are probably stressed. Such experiments are in progress to try to quantify the stress introduced by cross-fostering.
1091 Following a stress of the mother, the animals needed a long time to explore their environment. Indeed, the number of animals that explored the lighted compartment of the shuttle box for a long time was significantly higher than that of controls. Such a low activity and a low level of exploration behavior were also noted previously (1,32). Moreover, spatial memory was altered on a one-trial learning experiment: in the delayed alternation task, the experimental animals, whatever the group, had forgotten the arm visited 1 h before and had partially forgotten the arm visited 3 min before. Finally, the passive avoidance conditioning test also demonstrated a partial alteration of memory in the experimental groups. These results suggest that an acute stress at Day 10 or 19 of gestation in the rat influences the development of cognitive functions of the offspring, but one can only speculate about such effects. It is possible that the stresses generated by electric footshocks may act on the fetuses directly, but this can hardly be the case for the cat stress, which is exclusively emotional in nature. It seems more likely that the alterations of the cognitive functions were due to delayed growth rate (at least in the offspring of mothers stressed by the cat) as well as to deficits in maternal care and/or to subtle alterations in the development of the nervous system through modifications of the hormonal components of the mothers. It has already been said that a proper mother–fetus hormonal balance is needed for the development of the fetuses and it has also been suggested that stress during pregnancy might be a predisposing factor to mental illness (14,17) and to increased depression-related behavior in the female offspring during adulthood (2). Experiments are intended to try to correlate cognitive defects of young rats with modifications of the mother–fetus hormonal balance and with eventual alterations of their nervous system biochemistry.
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LORDI ET AL. 30. Stott, D. H. Follow-up study from birth of the effects of prenatal stresses. Dev. Med. Child Neurol. 15:770 –787; 1973. 31. Stott, D. H.; Camb, M. A.; Lond, P. D. Physical and mental handicaps following a disturbed pregnancy. Lancet 18:1006 –1012; 1957. 32. Thompson, W. R. Influence of prenatal maternal anxiety on emotionality in young rats. Science 125:698 – 699; 1957. 33. Thompson, W. D., Jr.; Sontag, L. W. Behavioral effects in the offspring of rats subjected to audiogenic seizures during the gestational period. J. Comp. Physiol. Psychol. 49:454 – 456; 1956. 34. Weir, M. W.; De Fries, J. C. Blocking of pregnancy in mice as a function of stress. Psychol. Reprod. 13:365–366; 1963. 35. Wildt, D. E.; Riegle, G. D.; Dukelow, W. R. Physiological temperature response and embryonic mortality in stressed swine. Am. J. Physiol. 229:1471–1475; 1975. 36. Zondek, B.; Tamari, I. Effects of auditory stimuli on reproduction. Ciba Found. Study Group 26:4 –19; 1967.