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Effects of changes in maternal pituitary-adrenal function on behavior of rat offspring
Physiology and Behavior, Vol. 8, pp. 425--430. Brain Research Publications Inc., 1972. Printed in Great Britain
Effects of Changes in Maternal Pituitary-Adrenal Function on Behavior of Rat Offspring' J. M. J O F F E ~-, K. M1LKOVI(~ ~, A N D S E Y M O U R L E V I N E '
Department of Psychiatry, Stanford University, School of Medicine, StanJord, CaliJbrnia 94305, U.S.A. (Received 11 June 1971) J. M., K. MILKOVI6AND S. LEVINE. Effects of changes in maternal p#uitary-adrenal function on behavior of rat offspring. PHYSIOL.BEHAV.8 (3) 425--430, 1972.--Pituitary-adrenal function of pregnant rats was altered by adrenalectoJOFFE,
mizing females prior to mating (Adx) or implanting ACTH secreting tumor tissue (MtT) subcutaneously. Their offspring were delivered by caesarean section and fostered to untreated females. Control groups were offspring of untreated females delivered by caesarean section and fostered to intact mothers, and normally delivered offspring fostered to untreated mothers. Offspring were tested from 60 days of age in an avoidance conditioning task and in an open field. Avoidance scores of females were increased by maternal adrenalectomy, while scores of males were not significantly affected by any of the prenatal treatments. Overall open-field activity was increased by both prenatal treatments and by caesarean delivery alone, but this outcome was dependent on a different pattern of scores in the Adx and MtT groups as compared to the caesarean-delivered controls. The activity scores were differentially affected by an interaction of the prenatal treatments with test-order and test trials. Pituitary Adrenal ACTH Corticosterone Offspring behavior Maternal endocrine function
Prenatal
Open-field activity
Avoidance conditioning
neonatal rat's neuroendocrine response to postnatal stimulation may affect its later behavior by producing a different setting of the regulatory mechanisms for H H A activity [12], and prenatal intervention may produce similar neonatal alteration of neuroendocrine organization. However, three studies in which effects of altered maternal pituitary-adrenal function on offspring behavior have been examined have reported no behavioral changes. Obias [20] found no effects of maternal hypophysectomy on Day 13 of gestation on the behavior of rat offspring, and Havlena and Werboff [8] found no behavioral effects in offspring of female rats adrenalectomized on Day 10½ or Day 16½ of gestation. More recently, Grota [6] found that adult offspring of pregnant rats which received up to 250 tzg of dexamethasone in their drinking water from Day 2 to Day 17 of gestation were not different from control offspring on a reaction-to-handling test though the highest dose of prenatal dexamethasone affected the resting level of plasma corticosterone in male offspring in adulthood. Grota [6] found a greater plasma corticosterone response to stimulation in 100 day old rats which had been reared to weaning by mothers which received dexamethasone during lactation, The purpose of the present study was to investigate the behavioral effects of prenatal changes in the maternal H H A
EXI'~RIMENTALmanipulation of the hypothalamo-hypophysealadrenocortical (HHA) system of the pregnant female rat affects the size and function of the foetal adrenals. Maternal adrenalectomy results in hypertrophied adrenals [9, 13, 16] and higher levels of plasma corticosterone [21] in the offspring at birth. Corticoid [2, 3, 19] or A C T H [15, 18] injections, on the other hand, result in smaller adrenals in the offspring. Similar inhibition of foetal adrenal growth can be achieved by transplantation of tissue from a tumor (mammatrophic tumor: MtT), which secretes ACTH, prolactin, and growth hormone, to the pregnant female rat [14]. There is little evidence on the effects of prenatal alteration of maternal H H A function on the behavior of the offspring, though such effects might be predicted for the following reasons. Alterations of the maternal H H A system produce changes in the pituitary-adrenocortical function of the neonatal animal. Since manipulations of the immature organism postnatally (such as neonatal handling or shock) which are known to affect profoundly the development of rodents [10] also alter the basal and stress levels of plasma corticosterone [5], one might expect the prenatal alterations of the matemal H H A system with their consequences for the neonatal function of the pituitary-adrenocortical system of the offspring, to affect later offspring behavior. The
1This study was supported by Research Grant NICH and HD 02881 from the National Institute of Health. We thank Dr. Steven Goldstein for advice on the statistical analyses. zSupported by Biological Sciences Training Grant MH 8304-06. Present address: Department of Psychology, University of Vermont, Burlington, Vermont, 05401, U.S.A. 3Supported by Grant G68-407, Foundations' Fund for Research in Psychiatry, New Haven, Connecticut, U.S.A. Present address: Biological Institute, Medical Faculty, University, Zagreb, Yugoslavia. 4Supported by USPHS Research Scientist Award 1-K05-MH-19936 from the National Institute of Mental Health. 425
426
JOFFE, MILKOV1C AND LEVINE
system by examining the effects on offspring of both increased and decreased levels of maternal corticosteroids.
METHOD
Subjects Rats of the Fischer strain were obtained from a local supplier. They were 90±5 days old on arrival in the laboratory.
Apparatus The offspring were tested in two situations. (a) In one of Ibur automated shuttleboxes which have been previously described [11]. The CS was a white noise accompanied by an increase in illumination in the compartment occupied by the rat; the UCS was a scrambled AC shock of 0.80mA delivered to the grid floor of the shuttlebox. (b) In a standardized open field similar to that described by Broadhurst [1]. Briefly, it consisted of a circular arena 32[ in. in dia. with a white painted plywood wall and floor. The floor is marked by three concentric circles and divided into segments, each approximately an equal distance across, by sections of radii. Illumination was provided by three 150 W bulbs in aluminum holders approximately 4 ft above floor level and averaged about 160ft-c at floor level. A loudspeaker positioned 4 ft above the center of the field provided white noise with an intensity of 77-79 db (ref. 0.0002 dyne/cm 2) at floor level; this provides a masking noise for extraneous sounds and increases the stressfulness of the situation.
Procedure After arrival the rats were numbered by ear-punching and then handled daily for a week prior to the experiment. One group of females was adrenalectomized (group Adx) under ether anesthesia at this point, following which the females were maintained on 0.9% saline in place of tap water. Adrenalectomy was carried out prior to mating to avoid stress induced by the procedure during pregnancy. Three days after surgery these animals as well as all the others used in the experiment were mated: males were placed in the females' cages every night and by 9 a.m. the following morning vaginal smears were taken and examined microscopically for the presence of sperm. The day when a positive smear was detected was considered the first day of pregnancy. On the first day of pregnancy a group of pregnant females (Group MtT) were anesthetized with ether and small pieces of tumor tissue were implanted under the skin of the hind legs according to the procedure described by Milkovi6 et al. [15, 16]. The rest of the pregnant females were left undisturbed until the 22nd day of pregnancy. On day 22 of gestation, that is, immediately prior to birth, the foetuses in the Adx and MtT groups as well as one group of intact females (control caesarean group) were delivered by a rapidly performed caesarean section under ether anesthesia. Caesarean-delivered pups were washed carefully with lukewarm water, warmed on an electric heating pad, and then fostered to intact females which had delivered their own pups not more than 24 hr earlier; the litters of these foster-mothers were removed immediately prior to fostering. Pups were delivered by caesarean section since pregnant
tbmales with well developed tumors are unable to deliver spontaneously. One group of intact tbmales delivered litters normally. Their pups were fostered within 24 hr of birth to other intact females which had delivered their litters within the previous 24 hr. This group of offspring (group control-fostered) was included to provide a comparison for the caesarean-delivered offspring of intact females and thus to assess the effects of caesarean section per se. Two pups were removed from each litter immediately after delivery, weighed, and their adrenals removed and weighed. The remaining pups in the four groups (Adx, MtT, ControlCaesarean, Control normal-delivery fostered) were reared by their foster-mothers until they were 21 days old. During that period litters were undisturbed. At 21 days offspring were weaned, weighed, and ear-punched. Six animals of the same sex and group were caged together and maintained under standard laboratory conditions (temperature 25~C; light-dark: 12 h r-12 hr). When the offspring were two months old, two males and two females were selected from each litter, with the animals matched for body weight at weaning. Each group consisted of a total of 20 animals (10 males and 10 females), i.e., 4 animals from each of 5 litters. Each group was then divided randomly into two subgroups, A and B, containing one male and one female from each litter, a total of 10 animals per subgroup. After this the animals were subjected to the following testing schedule. Subgroup A of each group was first tested in a shuttle box for avoidance conditioning. Six days later the animals were tested in the open field on 4 consecutive days; testing was always carried out at the same time of the day, between 1 and 3 p.m. Subgroup B was first tested in the open field in the same way and then in the shuttle box. The division into subgroups was made in order to examine the effects of test order on behavior. The testing procedures were as follows. (a) Ninety-trial discriminated avoidance conditioning: after a 2-rain adaptation period the CS was presented for 10 trials. On each trial the CS was terminated by a shuttle response or after 8 sec. The intertrial interval was the same as that used in conditioning trials (mean 45 sec, range 30-60see randomly ordered). Following the pretest trials each animal was given 90 avoidance conditioning trials (8 sec CS followed by shock which was terminated by the animal's crossing to the opposite compartment. Responding with a latency of less than 8 sec from CS onset enabled the rat to avoid shock). (b) Animals were given four daily 2-rain trials in the open field. Number of fecal boluses deposited and number of floor segments traversed were recorded.
RESULTS
Avoidance Cbnditioning Data were analyzed (analysis of variance) as a 4 (treatments) × 2 (sexes)~ 2 (test orders) factorial design with five animals per cell. There was one significant effect, the treatment by sex interaction (F3,~4=: 3.36, p < 0.05). The relevant means are shown in Fig. 1. The analyses of variance for the effects of treatments on males and females separately indicated that prenatal treatments did not significantly affect avoidance conditioning in males (F3,64--- 1.37, p > 0.25), but did significantly affect
MATERNAL PITUITARY-ADRENAL AND OFFSPRING BEHAVIOR the scores of females (F3,6, ~- 8.72, p < 0.01). A NewmanKeuls test for the significance of the differences between pairs of mean scores (females) indicated that the scores of MtT, Caesarean, and Control-Fostered females did not differ significantly (p > 0.05) but that Adx females had a significantly higher avoidance score than MtT or controlfostered subjects (p < 0.05); the Adx mean is not significantly higher than that of the caesarean females.
Open-yield Defecation and Activity Each of these sets of data were analyzed as a 4 (treatments) x 2 (sexes) x 2 (test orders) × 4 (test trials: repeated measure) factorial design with five animals per cell. None of these variables significantly affected open-field defecation although MtT offspring ( X - - 5.75 boluses per animal over four days of testing) defecated slightly less than offspring in the other groups (Means: Adx--9.00; C o n t r o l - C a e s a r e a n 7.90; Control-Fostered--8.70); this difference between MtT
427
offspring and the others is just short of significance (F3,e4 ~ 2.68; a t p = 0.05, F = 2.75). The analysis of activity scores revealed a number of significant effects including a significant interaction of treatment with test order and days of testing. This interaction qualifies the main effects and lower order interactions which are consequently not discussed separately. The significant effects found in the initial analysis of variance and the analyses of the lower order interactions and simple effects involved in the three-way interaction are summarized in Table 1. The mean scores of the groups involved in the interaction are shown in Fig. 2. The complexity of the interaction precludes a simple description of it, so only the manner in which it relates to significant main effects and lower order interactions will be described. The order by days effect is seen only in the MtT and Adx groups: animals in these two groups tested in the open field before being tested in the shuttle box (order B) were significantly more active (see Table 1) in the field on Day 1 than MtT and Adx animals
TABLE 1 SUMMARY OF ANALYSESOF VARIANCE: OPEN-HELD ACHVlTY SCORES
Source of Variation*
Sum of Squares
df
Between Subjects Treatment (T) Rats within groups
2178.66 16758.20
3 64
726.22 261.84
2.77?
Within Subjects Days (D) Order (O) x D T x O x D D x Rats within groups
33991.27 1402.91 2007.42 19640.20
3 3 9 192
11330.42 467.64 223.05 102.29
110.76++ 4.57++ 2.18?
400.57 476.02 518.54 646.82 46.18 141.21
2.81t 3.34t 3.64t 4.54++ <1 -< 1
352.65
2.48
590.96
4.15++
576.23 253.36 409.49 16.21 75.53 392.31 8.62 686.03 142.18
4.05++ 1.78 2.88t <1 <- 1 2.75t <1 4.82++
Interactions and Simple Effects in T x O x D Interaction O × D for Treatments MtT 1201.71 3 Day 1: OA vs. OB 1428.05 1 Adx 1555.64 3 Day 1: OA vs. Oa 1940.45 ! Caesarean 138.55 3 Control-Fostered 423.64 3 T x D for Order Order A 1057.97 3 [Significant Days effect (p < 0.01) for all treatments] Order B 1772.90 3 [Significant Days effect (p < 0.01) for all treatments] T x OforDays Day 1 O A: Treatments O a: Treatments Day 2 Day 3 Day 4 OA : Treatments OB: Treatments Error (within cells)
1728.71 760.08 1228.48 48.64 226.60 1176.94 25.88 2058.10 36398.40
3 3 3 3 3 3 3 3 256
*All excluded terms had non-significant F-values (p > 0.05). tP < 0.05. ++p< 0.01.
Mean Square
F
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JOFFE, MILKOVIC AND LEVINE
FIGURE
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over the four days of testing were shown by all subgroups, as might be expected from the F-value associated with days. The effects of the prenatal treatments on activity are found on Days 1 and 4, with no significant differences between treatment groups on Days 2 and 3; on Days 1 and 4 the differences between treatment groups are found only among the groups tested in the open field first (order B). In considering Day 1, order B scores it is found that Adx animals had significantly higher scores (p < 0.05, Newman-Keuls test) than Control-Caesarean or Control-Fostered offspring; MtT animals did not differ significantly from either Adx animals or controls and the two control groups' means were not significantly different. The means for the treatment groups on Day I (order B) were: MtT--49.1; Adx--53.8; Control-Caesarean--41.4; Control-Fostered--40.4. On Day 4, MtT animals, which were the only ones to show an increase in activity on the last day of testing, had a significantly higher score than all other order B subgroups (Newman-Keuls test, p < 0.05), which did not differ significantlyamong themselves. On the overall activity scores (see Table 1) the ControlFostered group ( ~ ~ 82.3) was significantly lower (NewmanKeuls test, p < 0.01 in all cases) than MtT ( ~ ~ 103.3), Adx(£~-- 110,4), and Control-Caesarean ( X - - 102.2) animals. The latter three groups' means were not significantly different.
GROUP
FIG. l. Number of avoidances of male and female offspring in the four treatment groups (Mean ~standard error).
with prior experience in the shuttle box (order A), but on subsequent days there were no significant differences between test orders for these subjects. Caesarean and Control-Foster offspring were not affected by test order. Although the pattern differs, significant changes in activity
DISCUSSION
The effects on offspring behavior of treatments intended primarily to alter maternal corticosteroid levels are complex and by no means consistent with a simple explanation in terms of slowed (MtT) or enhanced (Adx) foetal adrenal growth and development. The avoidance conditioning data show an interaction of prenatal treatment with sex of offspring: males were not significantly affected by any of the
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FIG. 2. Mean daily openfield activity of offspring. A. Avoidance conditioning prior to open-field testing. B. No prior avoidance conditioning.
MATERNAL PITUITARY-ADRENAL AND OFFSPRING BEHAVIOR treatments whereas the scores of Adx females were significantly better relative to Control-Fostered and MtT offspring. A variable which may be of importance is caesarean delivery. The Adx females' avoidance scores were not significantly different from those of Caesarean females and the latter had non-significantly higher avoidance scores than Control-Fostered or MtT females. In any case the behavioral effects of caesarean delivery would appear to require further investigation since this group differed from the normally delivered controls in open-field activity. The effects of the prenatal treatments on open-field activity are much more complex. The overall activity of offspring was increased by both prenatal treatments and by caesarean delivery alone to approximately the same degree. Grota et al. [7] found that caesarean delivery did not affect open field activity of rats at weaning, but the present data indicate an effect on open-field activity at maturity. However, in the Control-Caesarean group the pattern of the scores over days, and depending on test order, is markedly different from the MtT and Adx animals, and when this pattern is examined the Control-Caesarean group resembles the Control-Fostered group more than it resembles the MtT or Adx groups. Both the control groups show little effect of test order and the decline in activity scores over days is similar, though less marked in the Control-Caesarean offspring. On the other hand the MtT and Adx groups were clearly affected by order of testing and in a similar manner: prior experience on the avoidance conditioning task (order A) resulted in significantly lower activity on Day 1 of open-field testing in comparison to scores of MtT and Adx animals tested in the open field prior to shuttle box testing. It seems reasonable to surmise that this may indicate that MtT and Adx offspring were more fearful or reactive and thus their open-field activity was depressed by the prior association of removal from the home cage with the stressful avoidance conditioning situation. This supposition is supported by the pattern shown by the subgroups tested in the open field prior to avoidance conditioning (Fig. 2B): a high activity score on D a y 1 of testing has been found to indicate high emotionality [4, 22, 23]. The avoidance conditioning data are ambiguous on this point: MtT and Adx males had lower mean scores than either of the control groups but this difference was not significant. As far as the females' data were concerned, the scores of the two groups which were more emotional by an open-field activity criterion (MtT and Adx) were affected in opposite directions. There is little indication in the present activity data of any difference in the effects of a procedure which raised maternal cortieosteroids (MtT) and one which lowered them (Adx), except for the significantly higher activity of MtT order B animals on Day 4 of testing. The similarity of the effects of maternal tumor implantation and adrenalectomy on open-field activity are of some interest.
429
Data on the effects of these treatments on neonatal adrenal size indicate that adrenal weight was significantly higher in Adx offspring (54.064-1.58mg/100g body weight) and significantly lower in MtT animals (19.57-4- 0.83 mg/100 g b.w.), as compared to offspring of controls (42.61 + 1.26rag/100 g b.w.). However, in adulthood (70 days) both groups of offspring (MtT--25.63 +0.96; Adx--25.88 4-1.09) had significantly smaller adrenals than control-fostered offspring (30.61+0.62). Neonatal adrenal size does not provide an index of level of circulating plasma corticosterone (and this level may be the critical variable in terms of enduring effects on central neural organization). Although adrenalectomy and the mammatrophic tumor produce opposite effects on foetal adrenal size this may be irrelevant, and plasma corticosteroid levels are very probably high in both cases: in the foetus of an adrenalectomized female the foetal adrenal hypertrophy suggests increased steroid output, whereas in the foetus of an MtT female, where the foetal adrenals are atrophied, the circulating level of steroids (of maternal origin) will also be high, but these steroids will depress foetal A C ~ H output and produce the consequent foetal adrenal atrophy. (At term, MtT females' adrenals weighed 114.27 ± 6.02 mg/100 g body weight compared to a figure of 22.05+ 0.63 for control females.) This question is partly one of how and when the effects occur; there could be direct effects of changes in the maternal pituitary-adrenal system on foetal brain organization or there could be effects of the mother on the foetal pituitary-adrenal system which then produces effects on brain organization in the offspring, either prenatally or postnatally. However, the adrenal corticosterone responsiveness to stress in the two groups (Adx and MtT) was dissimilar in adulthood despite the similarity of adrenal size so that speculation about the neonatal levels of corticosterone and the possible implications of these levels must await experimental investigation. In addition, it would be premature to try to implicate the pituitary-adrenocortical system alone: the dissimilarity of the relative positions of the groups on two behavioral indices, open-field activity and avoidance conditioning, suggests the possibility that other aspects of physiological function have been altered by the prenatal treatments. It is of course possible that the pituitary-adrenocortical changes are only a part of the physiological substrate affected by the treatments. Generally, it is clear that prenatal alteration of the maternal hypothalamo-hypophyseal-adrenocortical system can affect a number of aspects of offspring behavior. This points to the possible influence of pituitary-adrenal hormones in the perinatal period on neuroendocrine substrates which affect later behavior, but considerable experimentation is still required to clarify the details of the organizational processes and the behavioral effects.
REFERENCES
1. Broadhurst, P. L. Experiments in psychogenetics: applications of biometrical genetics to behavior. In: Experiments in personality, edited by H. J. Eysenck, Vol. 1: Psychogenetics andpsychopharmacology. London: Routledge and Kegan Paul, 1960, pp. 1-102. 2. Courtier, R., A. Colonge and M. Baelesse. Action de la cortisone administr~e/t la m6re sur la surr6n~de du foetus de rat. C. R. Acad. Sci. 233: 333-336, 1951.
3. Davis, M. E. and E. J. Plotz. Effects of cortisone acetate on intact and adrenalectomized rats during pregnancy. Endocrinology 54: 384--395, 1954. 4. Denenberg, V. H. Open-field behavior in the rat: what does it mean? Ann. N.Y. Acad. Sci. 159: 852-859, 1969. 5. Denenberg, V. H., J. T. Brumaghim, G. C. Haltmeyer and M. X. Zarrow. Increased adrenocortical activity in the neonatal rat following handling. Endocrinology 81: 1047-1052, 1967.
430 6. Grota, L. J. Effects of dexamethasone on the development of emotionality and adrenocortical reactivity. Proc. 78th Ann. Cony. Am. psychol. Ass. 203-204, 1970. 7. Grota, L. J., V. H. Denenberg and M. X. Zarrow. Normal versus caesarean delivery: effects upon survival probability, weaning weight, and open-field activity. J. comp. physiol. Psychol. 61: 159-160, 1966. 8. Havlena, J. and J. Werboff. Adrenalectomy of the pregnant rat and behavior of the offspring. Psychol. Rep. 12: 348-350, 1963. 9. Houssay, B. A. Action de la insuficiencia suprarenal durante la prenez sobre la madre y el hijo. Rev. Soc. Argent. Biol. 21 : 316-331, 1945. 10. Levine, S. The psychophysiological effects of infantile stimulation. In: Roots of Behavior, edited by E. L. Bliss. New York: Hoeber, 1962. 11. Levine, S, UCS intensity and avoidance learning. J. exp. Psychol. 71: 163-164, 1966. 12. Levine, S. and R. F. Mullins, Jr. Hormonal influence on brain organization in infant rats. Science 152:1585-1592, 1966. 13. Milkovi6, K. and S. MilkoviC The influence of adrenalectomy of pregnant rats on the reactiveness of the pituitary-adrenal system of the newborn animals. Arch hltern. Physiol. Bioehem. 67: 25-28, 1959. 14. Milkovi6, K. and S. Milkovi6. Adrenocorticotrophic hormone secretion in the fetus and infant. In: Neuroendocrinology, edited by L. Martini and W. F. Ganong. New York: Academic Press, 1966, Vol. 1, p. 371. 15. Milkovi6, K., S. Efendi6, J. Paunovi6, J. Ronkulin, V. Dulibi¢~ and S. Milkovi6. Effect of the autonomous transplantable
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16. 17. 18.
19. 20. 21. 22.
23.
pituitary mammatrophic tumour on the pregnancy and the embryonic development in rat. Bull. scient. Cons. Acads RPF Yougosl., Section A--Zagreb 11: 107-108, 1966. Milkovid, S. and K. Milkovi6. Development of response to stress. Prog. Endocrinol. 184: 1077-1081, 1968. Milkovi6, S., K. Milkovi6 and S. Efendi6. Significance of maternal corticosteroids in fetal adrenal development and organogenesis. Gen. comp. Endoer. 10: 240-245, 1968. Milkovi6, S., J. Paunovi6, J. Ronkulin, T. Bobanovi6 and K. Milkovid. Some studies of the placental permeability to ACTH. Bull. scient., Cons. Acads RPF Yougosl., Section A - Zagreb 11: 108-109, 1966. Noumura, T. Development of the hypophyseal-adrenocortical system in the rat embryo in relation to the maternal system. Jap. J. Zool. 12: 279-299, 1959. Obias, M. D. Maternal behavior of hypophysectomized gravid albino rats and the development and performance of their progeny. J. comp. physiol. Psychol. 50: 120-124, 1957. Thoman, E., M. Sproul and S. Levine. Influence of adrenalectomy in female rats on reproductive processes: effects on the foetus and offspring. J. Endocrhtol. 46: 297-303, 1970. Whimbey, A. E. and V. H. Denenberg. Experimental programming of life histories: the factor structure underlying experimentally created individual differences. Behaviour 29: 296-314, 1967. Whimbey, A. E. and V. H. Denenberg. Two independent dinaensions in open-field performance. J. comp. physiol. Psyehol. 63:500 504, 1967.
Effects of Changes in Maternal Pituitary-Adrenal Function on Behavior of Rat Offspring' J. M. J O F F E ~-, K. M1LKOVI(~ ~, A N D S E Y M O U R L E V I N E '
Department of Psychiatry, Stanford University, School of Medicine, StanJord, CaliJbrnia 94305, U.S.A. (Received 11 June 1971) J. M., K. MILKOVI6AND S. LEVINE. Effects of changes in maternal p#uitary-adrenal function on behavior of rat offspring. PHYSIOL.BEHAV.8 (3) 425--430, 1972.--Pituitary-adrenal function of pregnant rats was altered by adrenalectoJOFFE,
mizing females prior to mating (Adx) or implanting ACTH secreting tumor tissue (MtT) subcutaneously. Their offspring were delivered by caesarean section and fostered to untreated females. Control groups were offspring of untreated females delivered by caesarean section and fostered to intact mothers, and normally delivered offspring fostered to untreated mothers. Offspring were tested from 60 days of age in an avoidance conditioning task and in an open field. Avoidance scores of females were increased by maternal adrenalectomy, while scores of males were not significantly affected by any of the prenatal treatments. Overall open-field activity was increased by both prenatal treatments and by caesarean delivery alone, but this outcome was dependent on a different pattern of scores in the Adx and MtT groups as compared to the caesarean-delivered controls. The activity scores were differentially affected by an interaction of the prenatal treatments with test-order and test trials. Pituitary Adrenal ACTH Corticosterone Offspring behavior Maternal endocrine function
Prenatal
Open-field activity
Avoidance conditioning
neonatal rat's neuroendocrine response to postnatal stimulation may affect its later behavior by producing a different setting of the regulatory mechanisms for H H A activity [12], and prenatal intervention may produce similar neonatal alteration of neuroendocrine organization. However, three studies in which effects of altered maternal pituitary-adrenal function on offspring behavior have been examined have reported no behavioral changes. Obias [20] found no effects of maternal hypophysectomy on Day 13 of gestation on the behavior of rat offspring, and Havlena and Werboff [8] found no behavioral effects in offspring of female rats adrenalectomized on Day 10½ or Day 16½ of gestation. More recently, Grota [6] found that adult offspring of pregnant rats which received up to 250 tzg of dexamethasone in their drinking water from Day 2 to Day 17 of gestation were not different from control offspring on a reaction-to-handling test though the highest dose of prenatal dexamethasone affected the resting level of plasma corticosterone in male offspring in adulthood. Grota [6] found a greater plasma corticosterone response to stimulation in 100 day old rats which had been reared to weaning by mothers which received dexamethasone during lactation, The purpose of the present study was to investigate the behavioral effects of prenatal changes in the maternal H H A
EXI'~RIMENTALmanipulation of the hypothalamo-hypophysealadrenocortical (HHA) system of the pregnant female rat affects the size and function of the foetal adrenals. Maternal adrenalectomy results in hypertrophied adrenals [9, 13, 16] and higher levels of plasma corticosterone [21] in the offspring at birth. Corticoid [2, 3, 19] or A C T H [15, 18] injections, on the other hand, result in smaller adrenals in the offspring. Similar inhibition of foetal adrenal growth can be achieved by transplantation of tissue from a tumor (mammatrophic tumor: MtT), which secretes ACTH, prolactin, and growth hormone, to the pregnant female rat [14]. There is little evidence on the effects of prenatal alteration of maternal H H A function on the behavior of the offspring, though such effects might be predicted for the following reasons. Alterations of the maternal H H A system produce changes in the pituitary-adrenocortical function of the neonatal animal. Since manipulations of the immature organism postnatally (such as neonatal handling or shock) which are known to affect profoundly the development of rodents [10] also alter the basal and stress levels of plasma corticosterone [5], one might expect the prenatal alterations of the matemal H H A system with their consequences for the neonatal function of the pituitary-adrenocortical system of the offspring, to affect later offspring behavior. The
1This study was supported by Research Grant NICH and HD 02881 from the National Institute of Health. We thank Dr. Steven Goldstein for advice on the statistical analyses. zSupported by Biological Sciences Training Grant MH 8304-06. Present address: Department of Psychology, University of Vermont, Burlington, Vermont, 05401, U.S.A. 3Supported by Grant G68-407, Foundations' Fund for Research in Psychiatry, New Haven, Connecticut, U.S.A. Present address: Biological Institute, Medical Faculty, University, Zagreb, Yugoslavia. 4Supported by USPHS Research Scientist Award 1-K05-MH-19936 from the National Institute of Mental Health. 425
426
JOFFE, MILKOV1C AND LEVINE
system by examining the effects on offspring of both increased and decreased levels of maternal corticosteroids.
METHOD
Subjects Rats of the Fischer strain were obtained from a local supplier. They were 90±5 days old on arrival in the laboratory.
Apparatus The offspring were tested in two situations. (a) In one of Ibur automated shuttleboxes which have been previously described [11]. The CS was a white noise accompanied by an increase in illumination in the compartment occupied by the rat; the UCS was a scrambled AC shock of 0.80mA delivered to the grid floor of the shuttlebox. (b) In a standardized open field similar to that described by Broadhurst [1]. Briefly, it consisted of a circular arena 32[ in. in dia. with a white painted plywood wall and floor. The floor is marked by three concentric circles and divided into segments, each approximately an equal distance across, by sections of radii. Illumination was provided by three 150 W bulbs in aluminum holders approximately 4 ft above floor level and averaged about 160ft-c at floor level. A loudspeaker positioned 4 ft above the center of the field provided white noise with an intensity of 77-79 db (ref. 0.0002 dyne/cm 2) at floor level; this provides a masking noise for extraneous sounds and increases the stressfulness of the situation.
Procedure After arrival the rats were numbered by ear-punching and then handled daily for a week prior to the experiment. One group of females was adrenalectomized (group Adx) under ether anesthesia at this point, following which the females were maintained on 0.9% saline in place of tap water. Adrenalectomy was carried out prior to mating to avoid stress induced by the procedure during pregnancy. Three days after surgery these animals as well as all the others used in the experiment were mated: males were placed in the females' cages every night and by 9 a.m. the following morning vaginal smears were taken and examined microscopically for the presence of sperm. The day when a positive smear was detected was considered the first day of pregnancy. On the first day of pregnancy a group of pregnant females (Group MtT) were anesthetized with ether and small pieces of tumor tissue were implanted under the skin of the hind legs according to the procedure described by Milkovi6 et al. [15, 16]. The rest of the pregnant females were left undisturbed until the 22nd day of pregnancy. On day 22 of gestation, that is, immediately prior to birth, the foetuses in the Adx and MtT groups as well as one group of intact females (control caesarean group) were delivered by a rapidly performed caesarean section under ether anesthesia. Caesarean-delivered pups were washed carefully with lukewarm water, warmed on an electric heating pad, and then fostered to intact females which had delivered their own pups not more than 24 hr earlier; the litters of these foster-mothers were removed immediately prior to fostering. Pups were delivered by caesarean section since pregnant
tbmales with well developed tumors are unable to deliver spontaneously. One group of intact tbmales delivered litters normally. Their pups were fostered within 24 hr of birth to other intact females which had delivered their litters within the previous 24 hr. This group of offspring (group control-fostered) was included to provide a comparison for the caesarean-delivered offspring of intact females and thus to assess the effects of caesarean section per se. Two pups were removed from each litter immediately after delivery, weighed, and their adrenals removed and weighed. The remaining pups in the four groups (Adx, MtT, ControlCaesarean, Control normal-delivery fostered) were reared by their foster-mothers until they were 21 days old. During that period litters were undisturbed. At 21 days offspring were weaned, weighed, and ear-punched. Six animals of the same sex and group were caged together and maintained under standard laboratory conditions (temperature 25~C; light-dark: 12 h r-12 hr). When the offspring were two months old, two males and two females were selected from each litter, with the animals matched for body weight at weaning. Each group consisted of a total of 20 animals (10 males and 10 females), i.e., 4 animals from each of 5 litters. Each group was then divided randomly into two subgroups, A and B, containing one male and one female from each litter, a total of 10 animals per subgroup. After this the animals were subjected to the following testing schedule. Subgroup A of each group was first tested in a shuttle box for avoidance conditioning. Six days later the animals were tested in the open field on 4 consecutive days; testing was always carried out at the same time of the day, between 1 and 3 p.m. Subgroup B was first tested in the open field in the same way and then in the shuttle box. The division into subgroups was made in order to examine the effects of test order on behavior. The testing procedures were as follows. (a) Ninety-trial discriminated avoidance conditioning: after a 2-rain adaptation period the CS was presented for 10 trials. On each trial the CS was terminated by a shuttle response or after 8 sec. The intertrial interval was the same as that used in conditioning trials (mean 45 sec, range 30-60see randomly ordered). Following the pretest trials each animal was given 90 avoidance conditioning trials (8 sec CS followed by shock which was terminated by the animal's crossing to the opposite compartment. Responding with a latency of less than 8 sec from CS onset enabled the rat to avoid shock). (b) Animals were given four daily 2-rain trials in the open field. Number of fecal boluses deposited and number of floor segments traversed were recorded.
RESULTS
Avoidance Cbnditioning Data were analyzed (analysis of variance) as a 4 (treatments) × 2 (sexes)~ 2 (test orders) factorial design with five animals per cell. There was one significant effect, the treatment by sex interaction (F3,~4=: 3.36, p < 0.05). The relevant means are shown in Fig. 1. The analyses of variance for the effects of treatments on males and females separately indicated that prenatal treatments did not significantly affect avoidance conditioning in males (F3,64--- 1.37, p > 0.25), but did significantly affect
MATERNAL PITUITARY-ADRENAL AND OFFSPRING BEHAVIOR the scores of females (F3,6, ~- 8.72, p < 0.01). A NewmanKeuls test for the significance of the differences between pairs of mean scores (females) indicated that the scores of MtT, Caesarean, and Control-Fostered females did not differ significantly (p > 0.05) but that Adx females had a significantly higher avoidance score than MtT or controlfostered subjects (p < 0.05); the Adx mean is not significantly higher than that of the caesarean females.
Open-yield Defecation and Activity Each of these sets of data were analyzed as a 4 (treatments) x 2 (sexes) x 2 (test orders) × 4 (test trials: repeated measure) factorial design with five animals per cell. None of these variables significantly affected open-field defecation although MtT offspring ( X - - 5.75 boluses per animal over four days of testing) defecated slightly less than offspring in the other groups (Means: Adx--9.00; C o n t r o l - C a e s a r e a n 7.90; Control-Fostered--8.70); this difference between MtT
427
offspring and the others is just short of significance (F3,e4 ~ 2.68; a t p = 0.05, F = 2.75). The analysis of activity scores revealed a number of significant effects including a significant interaction of treatment with test order and days of testing. This interaction qualifies the main effects and lower order interactions which are consequently not discussed separately. The significant effects found in the initial analysis of variance and the analyses of the lower order interactions and simple effects involved in the three-way interaction are summarized in Table 1. The mean scores of the groups involved in the interaction are shown in Fig. 2. The complexity of the interaction precludes a simple description of it, so only the manner in which it relates to significant main effects and lower order interactions will be described. The order by days effect is seen only in the MtT and Adx groups: animals in these two groups tested in the open field before being tested in the shuttle box (order B) were significantly more active (see Table 1) in the field on Day 1 than MtT and Adx animals
TABLE 1 SUMMARY OF ANALYSESOF VARIANCE: OPEN-HELD ACHVlTY SCORES
Source of Variation*
Sum of Squares
df
Between Subjects Treatment (T) Rats within groups
2178.66 16758.20
3 64
726.22 261.84
2.77?
Within Subjects Days (D) Order (O) x D T x O x D D x Rats within groups
33991.27 1402.91 2007.42 19640.20
3 3 9 192
11330.42 467.64 223.05 102.29
110.76++ 4.57++ 2.18?
400.57 476.02 518.54 646.82 46.18 141.21
2.81t 3.34t 3.64t 4.54++ <1 -< 1
352.65
2.48
590.96
4.15++
576.23 253.36 409.49 16.21 75.53 392.31 8.62 686.03 142.18
4.05++ 1.78 2.88t <1 <- 1 2.75t <1 4.82++
Interactions and Simple Effects in T x O x D Interaction O × D for Treatments MtT 1201.71 3 Day 1: OA vs. OB 1428.05 1 Adx 1555.64 3 Day 1: OA vs. Oa 1940.45 ! Caesarean 138.55 3 Control-Fostered 423.64 3 T x D for Order Order A 1057.97 3 [Significant Days effect (p < 0.01) for all treatments] Order B 1772.90 3 [Significant Days effect (p < 0.01) for all treatments] T x OforDays Day 1 O A: Treatments O a: Treatments Day 2 Day 3 Day 4 OA : Treatments OB: Treatments Error (within cells)
1728.71 760.08 1228.48 48.64 226.60 1176.94 25.88 2058.10 36398.40
3 3 3 3 3 3 3 3 256
*All excluded terms had non-significant F-values (p > 0.05). tP < 0.05. ++p< 0.01.
Mean Square
F
428
JOFFE, MILKOVIC AND LEVINE
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over the four days of testing were shown by all subgroups, as might be expected from the F-value associated with days. The effects of the prenatal treatments on activity are found on Days 1 and 4, with no significant differences between treatment groups on Days 2 and 3; on Days 1 and 4 the differences between treatment groups are found only among the groups tested in the open field first (order B). In considering Day 1, order B scores it is found that Adx animals had significantly higher scores (p < 0.05, Newman-Keuls test) than Control-Caesarean or Control-Fostered offspring; MtT animals did not differ significantly from either Adx animals or controls and the two control groups' means were not significantly different. The means for the treatment groups on Day I (order B) were: MtT--49.1; Adx--53.8; Control-Caesarean--41.4; Control-Fostered--40.4. On Day 4, MtT animals, which were the only ones to show an increase in activity on the last day of testing, had a significantly higher score than all other order B subgroups (Newman-Keuls test, p < 0.05), which did not differ significantlyamong themselves. On the overall activity scores (see Table 1) the ControlFostered group ( ~ ~ 82.3) was significantly lower (NewmanKeuls test, p < 0.01 in all cases) than MtT ( ~ ~ 103.3), Adx(£~-- 110,4), and Control-Caesarean ( X - - 102.2) animals. The latter three groups' means were not significantly different.
GROUP
FIG. l. Number of avoidances of male and female offspring in the four treatment groups (Mean ~standard error).
with prior experience in the shuttle box (order A), but on subsequent days there were no significant differences between test orders for these subjects. Caesarean and Control-Foster offspring were not affected by test order. Although the pattern differs, significant changes in activity
DISCUSSION
The effects on offspring behavior of treatments intended primarily to alter maternal corticosteroid levels are complex and by no means consistent with a simple explanation in terms of slowed (MtT) or enhanced (Adx) foetal adrenal growth and development. The avoidance conditioning data show an interaction of prenatal treatment with sex of offspring: males were not significantly affected by any of the
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FIG. 2. Mean daily openfield activity of offspring. A. Avoidance conditioning prior to open-field testing. B. No prior avoidance conditioning.
MATERNAL PITUITARY-ADRENAL AND OFFSPRING BEHAVIOR treatments whereas the scores of Adx females were significantly better relative to Control-Fostered and MtT offspring. A variable which may be of importance is caesarean delivery. The Adx females' avoidance scores were not significantly different from those of Caesarean females and the latter had non-significantly higher avoidance scores than Control-Fostered or MtT females. In any case the behavioral effects of caesarean delivery would appear to require further investigation since this group differed from the normally delivered controls in open-field activity. The effects of the prenatal treatments on open-field activity are much more complex. The overall activity of offspring was increased by both prenatal treatments and by caesarean delivery alone to approximately the same degree. Grota et al. [7] found that caesarean delivery did not affect open field activity of rats at weaning, but the present data indicate an effect on open-field activity at maturity. However, in the Control-Caesarean group the pattern of the scores over days, and depending on test order, is markedly different from the MtT and Adx animals, and when this pattern is examined the Control-Caesarean group resembles the Control-Fostered group more than it resembles the MtT or Adx groups. Both the control groups show little effect of test order and the decline in activity scores over days is similar, though less marked in the Control-Caesarean offspring. On the other hand the MtT and Adx groups were clearly affected by order of testing and in a similar manner: prior experience on the avoidance conditioning task (order A) resulted in significantly lower activity on Day 1 of open-field testing in comparison to scores of MtT and Adx animals tested in the open field prior to shuttle box testing. It seems reasonable to surmise that this may indicate that MtT and Adx offspring were more fearful or reactive and thus their open-field activity was depressed by the prior association of removal from the home cage with the stressful avoidance conditioning situation. This supposition is supported by the pattern shown by the subgroups tested in the open field prior to avoidance conditioning (Fig. 2B): a high activity score on D a y 1 of testing has been found to indicate high emotionality [4, 22, 23]. The avoidance conditioning data are ambiguous on this point: MtT and Adx males had lower mean scores than either of the control groups but this difference was not significant. As far as the females' data were concerned, the scores of the two groups which were more emotional by an open-field activity criterion (MtT and Adx) were affected in opposite directions. There is little indication in the present activity data of any difference in the effects of a procedure which raised maternal cortieosteroids (MtT) and one which lowered them (Adx), except for the significantly higher activity of MtT order B animals on Day 4 of testing. The similarity of the effects of maternal tumor implantation and adrenalectomy on open-field activity are of some interest.
429
Data on the effects of these treatments on neonatal adrenal size indicate that adrenal weight was significantly higher in Adx offspring (54.064-1.58mg/100g body weight) and significantly lower in MtT animals (19.57-4- 0.83 mg/100 g b.w.), as compared to offspring of controls (42.61 + 1.26rag/100 g b.w.). However, in adulthood (70 days) both groups of offspring (MtT--25.63 +0.96; Adx--25.88 4-1.09) had significantly smaller adrenals than control-fostered offspring (30.61+0.62). Neonatal adrenal size does not provide an index of level of circulating plasma corticosterone (and this level may be the critical variable in terms of enduring effects on central neural organization). Although adrenalectomy and the mammatrophic tumor produce opposite effects on foetal adrenal size this may be irrelevant, and plasma corticosteroid levels are very probably high in both cases: in the foetus of an adrenalectomized female the foetal adrenal hypertrophy suggests increased steroid output, whereas in the foetus of an MtT female, where the foetal adrenals are atrophied, the circulating level of steroids (of maternal origin) will also be high, but these steroids will depress foetal A C ~ H output and produce the consequent foetal adrenal atrophy. (At term, MtT females' adrenals weighed 114.27 ± 6.02 mg/100 g body weight compared to a figure of 22.05+ 0.63 for control females.) This question is partly one of how and when the effects occur; there could be direct effects of changes in the maternal pituitary-adrenal system on foetal brain organization or there could be effects of the mother on the foetal pituitary-adrenal system which then produces effects on brain organization in the offspring, either prenatally or postnatally. However, the adrenal corticosterone responsiveness to stress in the two groups (Adx and MtT) was dissimilar in adulthood despite the similarity of adrenal size so that speculation about the neonatal levels of corticosterone and the possible implications of these levels must await experimental investigation. In addition, it would be premature to try to implicate the pituitary-adrenocortical system alone: the dissimilarity of the relative positions of the groups on two behavioral indices, open-field activity and avoidance conditioning, suggests the possibility that other aspects of physiological function have been altered by the prenatal treatments. It is of course possible that the pituitary-adrenocortical changes are only a part of the physiological substrate affected by the treatments. Generally, it is clear that prenatal alteration of the maternal hypothalamo-hypophyseal-adrenocortical system can affect a number of aspects of offspring behavior. This points to the possible influence of pituitary-adrenal hormones in the perinatal period on neuroendocrine substrates which affect later behavior, but considerable experimentation is still required to clarify the details of the organizational processes and the behavioral effects.
REFERENCES
1. Broadhurst, P. L. Experiments in psychogenetics: applications of biometrical genetics to behavior. In: Experiments in personality, edited by H. J. Eysenck, Vol. 1: Psychogenetics andpsychopharmacology. London: Routledge and Kegan Paul, 1960, pp. 1-102. 2. Courtier, R., A. Colonge and M. Baelesse. Action de la cortisone administr~e/t la m6re sur la surr6n~de du foetus de rat. C. R. Acad. Sci. 233: 333-336, 1951.
3. Davis, M. E. and E. J. Plotz. Effects of cortisone acetate on intact and adrenalectomized rats during pregnancy. Endocrinology 54: 384--395, 1954. 4. Denenberg, V. H. Open-field behavior in the rat: what does it mean? Ann. N.Y. Acad. Sci. 159: 852-859, 1969. 5. Denenberg, V. H., J. T. Brumaghim, G. C. Haltmeyer and M. X. Zarrow. Increased adrenocortical activity in the neonatal rat following handling. Endocrinology 81: 1047-1052, 1967.
430 6. Grota, L. J. Effects of dexamethasone on the development of emotionality and adrenocortical reactivity. Proc. 78th Ann. Cony. Am. psychol. Ass. 203-204, 1970. 7. Grota, L. J., V. H. Denenberg and M. X. Zarrow. Normal versus caesarean delivery: effects upon survival probability, weaning weight, and open-field activity. J. comp. physiol. Psychol. 61: 159-160, 1966. 8. Havlena, J. and J. Werboff. Adrenalectomy of the pregnant rat and behavior of the offspring. Psychol. Rep. 12: 348-350, 1963. 9. Houssay, B. A. Action de la insuficiencia suprarenal durante la prenez sobre la madre y el hijo. Rev. Soc. Argent. Biol. 21 : 316-331, 1945. 10. Levine, S. The psychophysiological effects of infantile stimulation. In: Roots of Behavior, edited by E. L. Bliss. New York: Hoeber, 1962. 11. Levine, S, UCS intensity and avoidance learning. J. exp. Psychol. 71: 163-164, 1966. 12. Levine, S. and R. F. Mullins, Jr. Hormonal influence on brain organization in infant rats. Science 152:1585-1592, 1966. 13. Milkovi6, K. and S. MilkoviC The influence of adrenalectomy of pregnant rats on the reactiveness of the pituitary-adrenal system of the newborn animals. Arch hltern. Physiol. Bioehem. 67: 25-28, 1959. 14. Milkovi6, K. and S. Milkovi6. Adrenocorticotrophic hormone secretion in the fetus and infant. In: Neuroendocrinology, edited by L. Martini and W. F. Ganong. New York: Academic Press, 1966, Vol. 1, p. 371. 15. Milkovi6, K., S. Efendi6, J. Paunovi6, J. Ronkulin, V. Dulibi¢~ and S. Milkovi6. Effect of the autonomous transplantable
JOFFE, MILKOVI(_" AND LEVINE
16. 17. 18.
19. 20. 21. 22.
23.
pituitary mammatrophic tumour on the pregnancy and the embryonic development in rat. Bull. scient. Cons. Acads RPF Yougosl., Section A--Zagreb 11: 107-108, 1966. Milkovid, S. and K. Milkovi6. Development of response to stress. Prog. Endocrinol. 184: 1077-1081, 1968. Milkovi6, S., K. Milkovi6 and S. Efendi6. Significance of maternal corticosteroids in fetal adrenal development and organogenesis. Gen. comp. Endoer. 10: 240-245, 1968. Milkovi6, S., J. Paunovi6, J. Ronkulin, T. Bobanovi6 and K. Milkovid. Some studies of the placental permeability to ACTH. Bull. scient., Cons. Acads RPF Yougosl., Section A - Zagreb 11: 108-109, 1966. Noumura, T. Development of the hypophyseal-adrenocortical system in the rat embryo in relation to the maternal system. Jap. J. Zool. 12: 279-299, 1959. Obias, M. D. Maternal behavior of hypophysectomized gravid albino rats and the development and performance of their progeny. J. comp. physiol. Psychol. 50: 120-124, 1957. Thoman, E., M. Sproul and S. Levine. Influence of adrenalectomy in female rats on reproductive processes: effects on the foetus and offspring. J. Endocrhtol. 46: 297-303, 1970. Whimbey, A. E. and V. H. Denenberg. Experimental programming of life histories: the factor structure underlying experimentally created individual differences. Behaviour 29: 296-314, 1967. Whimbey, A. E. and V. H. Denenberg. Two independent dinaensions in open-field performance. J. comp. physiol. Psyehol. 63:500 504, 1967.