Alcohol, Vol.9, pp. 219-223, 1992
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Prenatal Ethanol Effects: Sex Differences in Offspring Stress Responsiveness JOANNE W E I N B E R G
Department o f Anatomy, Faculty o f Medicine, The University o f British Columbia, Vancouver, BC V6T 1Z3 Canada Received 28 M a y 1991; Accepted 6 D e c e m b e r 1991 WEINBERG, J. Prenatal ethanol effects: Sex differences in offspring stress responsiveness. ALCOHOL 9(3) 219-223, 1992.-Previous studies have shown that offspring prenatally exposed to ethanol are hyperresponsive to stressors in adulthood, and have suggested that females are typically more affected than males. The present study was undertaken to investigate further this apparent sex difference in prenatal ethanol effects on stress responsiveness. Male and female offspring from prenatal ethanol-exposed (E), pair-fed (PF), and ad lib-fed control (C) conditions were tested in adulthood to determine adrenoconical responses to a prolonged (4-h) restraint stress. There were no significant differences in corticoid responsiveness among females from the three treatment groups. All females showed a marked increase in plasma corticosterone at 30 rain, and corticoid levels remained elevated through 150-min restraint. By 180 min, all females showed a significant corticoid decrease, although corticosterone remained elevated over basal levels throughout the 240-rain stress period. For males, in contrast, there were significant differences among groups. All males showed a significant corticoid increase over basal levels at 30 min, and corticoids remained significantly elevated through 90-min restraint. By 120 min, PF and C males showed a significant corticoid decrease although corticoids never returned to basal levels during the 240-min restraint period. E males, however, showed no significant decrease from peak corticosterone levels throughout the 240-min restraint stress. These data indicate that pituitary-adrenal hyperresponsiveness is not limited to fetal ethanol-exposed females, but may be demonstrated in fetal ethanol-exposed males under appropriate conditions. The parameters of the test situation, including the nature and intensity of the stressor and the time course of the response during stress or recovery from stress that is measured, appear to be critical in revealing differential effects of prenatal ethanol exposure on males and females. Fetal alcohol syndrome Sex differences Hyperresponsiveness to stress Rat
Stress
Pituitary-adrenal system
ETHANOL consumption by the pregnant female rat alters development and stress responsiveness of the offspring hypothalamo-pituitary-adrenal (HPA) axis. Fetal ethanolexposed (E) offspring have elevated brain and plasma corticosterone levels at birth (7,22,31), and show reduced adrenocortical responses to stressors and drug (e.g., ethanol, morphine) challenges during the preweaning period compared with PF and C offspring (25,31). /~-Endorphin (~-EP) responses to stressors are similarly suppressed in E compared with PF and C offspring prior to weaning (2). Interestingly, such reduced stress responsiveness is a transient phenomenon. Beginning in the period between weaning age and puberty, and persisting into adulthood, E animals often exhibit hyperresponsiveness to stressors compared with controls. Enhanced pituitaryadrenal activation to intermittent footshock, ether, psychological or neurogenic stressors, and drugs such as ethanol and morphine, as well as increased /~-EP responses to cold and ether stress have been reported in E compared with PF and C animals (2,14,23,24,27,30,32). In addition, E offspring may
also exhibit deficits in response inhibition or recovery of the pituitary-adrenal system toward basal levels following stress
(30). An important finding in these previous studies, at least with regard to the HPA axis, is that females appear to be more vulnerable than males to prenatal ethanol effects on stress responsiveness. Data from Taylor and coworkers (14, 23,24,27) demonstrating HPA hyperresponsiveness in E animals were derived primarily from females. Studies in our laboratory found deficits in adrenocortical response inhibition or recovery following stress only in E females; E males were similar in response to PF and C males (30). Similarly, a recent study by Kelley et al. (10) found that early postnatal ethanol exposure resulted in adrenocortical hyperresponsiveness in a forced swimming task in female but not male offspring. The present study was undertaken to investigate further this apparent sex difference in effects of prenatal ethanol exposure on stress responsiveness. Previous studies typically used stressors that were acute rather than chronic and/or of
Requests for reprints should be addressed to Dr. Joanne Weinberg, Department of Anatomy, The University of British Columbia, 2177 Wesbrook Mall, Vancouver, BC V6T IZ3, Canada. 219
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WEINBERG
relatively short duration. In the present study, adrenocortical responses to a prolonged s t r e s s o r - 4 - h r e s t r a i n t - w e r e examined. METHOD
Animals and Mating Forty Sprague-Dawley female rats (250-275 g; Canadian Breeding Farms, St. Constant, Quebec) were group housed in polycarbonate cages (46 x 24 × 15 cm) for a 1-2-week adaptation period. They were then housed singly in stainlesssteel suspended cages with wire mesh front and floor (25 x 18 × 18 cm) together with a Sprague-Dawley male (300-350 g), and cage papers were checked daily. The presence of a vaginal plug indicated day 1 of gestation. During adaptation and mating all animals had ad lib access to standard laboratory chow (Ralston Purina of Canada, Woodstock, Ontario) and water. The colony room had controlled temperature (2122°C) and lighting, with lights on 0600-1800 h.
Diets and Feeding On day 1 of gestation, females were randomly assigned to one of three groups: (1) ethanol (E), n = 14: liquid ethanol diet, ad lib; (2) pair-fed (PF), n = 13: liquid control diet, with each animal pair-fed the amount consumed by a female in the ethanol group (g/kg body weight per day of gestation); (3) control (C), n = 13: laboratory chow and water, ad lib. Diets were prepared by Bio-Serv, Inc. (Frenchtown, N J), and were formulated to provide adequate nutrition to pregnant females regardless of ethanol intake (29). The liquid ethanol diet contained 36% ethanol-derived calories; maltose-dextrin was isocalorically substituted for ethanol in the liquid control diet. Fresh diet was placed on the cages daily in the late afternoon (1-2-h prior to lights off); bottles from the previous day were removed and weighed at this time to determine amount consumed. This feeding schedule was designed to prevent a shift in the corticosterone circadian rhythm which typically occurs in animals on a restricted feeding schedule such as that of the pair-fed group (6). Females were weighed on days 1, 7, 14, and 21 of gestation. On the afternoon of day 22, experimental diets were replaced with laboratory chow and water. At birth (designated day 1 lactation), cages were cleaned, dam and pups were weighed, and all litters were culled to ten (five males, five females when possible). For litters of eight or nine pups, pups from a dam of the same treatment condition, born on the same day, were fostered in to bring the litter size to ten. Litters with fewer than eight pups were eliminated from the study. Other than once weekly weighing and cage cleaning, dam and pups remained undisturbed until weaning on day 22, after which offspring were group housed by litter and by sex until testing in adulthood.
Testing and Blood Sampling Testing began when animals were 80-90 days of age. No more than one male and one female from any litter were assigned to any one blood sampling time, and each animal was blood sampled only once in this study. Animals were individually housed for at least 1 week prior to testing. All testing was conducted between 0800-1300 h. On the test day, animals were assigned either to the basal or to the stress condition. Cages were quickly and quietly carried from the colony room
to an adjacent laboratory. For animals in the basal condition, blood samples (0.5 cc) were obtained immediately by cardiac puncture under light ether anesthesia. Animals in the stress condition were quickly removed from their cages and immediately confined to plastic (PVC) restraint tubes which restricted movement. Tubes used for males were 6.5 cm in diameter x 20.5 cm in length; tubes for females were 5 cm in diameter × 15.5 cm in length. At intervals throughout the 4-h restraint period, animals were removed from their restraint tubes and blood samples obtained by cardiac puncture as described above. Samples from males were taken at 30, 60, 90, 120, 180, and 240 min of restraint; samples from females were taken at 30, 60, 90, 150, 180, 210, and 240 min of restraint. The entire sampling procedure was completed within 2 min of touching the animal's home cage (for basal samples) or restraint tube (for stress samples), which is rapid enough to obtain a reliable measure of corticosterone without any effects of disturbance or of the procedure of etherization and cardiac puncture (4). The study was carried out over an approximately 4-week period; males and females from E, PF, and C conditions were represented on every test day.
Radioimmunoassay Plasma was extracted in absolute ethanol (1:10 vol/vol) and corticosterone measured by radioimmunoassay using our adaptation of the method of Kaneko et al. (8). Antiserum was obtained from Immunocorp (Montreal) PQ; tracer, [1,2,6,73H] corticosterone, was obtained from DuPont, Canada (NEN Research Products, Mississauga, Ontario) and unlabeled corticosterone was obtained from Sigma Chemical Co. (St. Louis, MO). Dextran-coated charcoal was used to absorb and precipitate free steroid after incubation. Samples were counted in Formula 989 (NEN Research Products). Total corticosterone (bound plus free) was determined by this method.
Statistical Analyses Data were analyzed by appropriate analyses of variance (ANOVAs) for the factors of group and time. Time was treated as a repeated measure for analysis of the developmental data and as a nonrepeated measure for analysis of corticosterone levels. Significant main or interaction effects were further analyzed by Newman-Keuls paired comparisons. ANOVA results are presented in the tables and figure legends. RESULTS
Developmental Data Ethanol intake of pregnant females was consistently high throughout gestation, averaging 9.6 +_ 0.28, 12.0 ± 0.28, and 11.6 ± 0.20 g/kg body weight for weeks l, 2, and 3 of gestation, respectively. We have shown previously that ethanol intake at these levels results in blood alcohol levels of approximately 145-155 mg/dl (29). A repeated measures ANOVA on maternal weight gain during pregnancy revealed significant effects of group (p < 0.01) and days (p < 0.001), as well as a significant group × days interaction (p < 0.001). Post hoc tests indicated that from gestation day 7 onward, E and PF females weighed less than C females (p's < 0.01) (day 21 data shown in Table 1). Similarly, E a n d P F females weighed less than C females on the day of birth (within 24-h postpartum) (p's < 0.05) (Table 1).
PRENATAL
STRESS AND STRESS RESPONSIVENESS
221
TABLE I PREGNANCY, BIRTH, AND DEVELOPMENTAL DATA FROM DAMS AND PUPS IN ETHANOL, PAIR-FED, AND CONTROL CONDITIONS Maternal Females
Pups Weaning Wt. (g)
E PF C
G21 B. Wt. (g)
Gestation Length (days)
350.0 + 6.3 (14) 352.0 ± 7.4 (13) 389.2 + 7.5 (12)*
23.36 ± 0.10 (14)t 22.96 _+ 0.09 (13) 23.04 ± 0.13 (13)
LIB. Wt. (g) 284.1 ± 2.7 (14) 292.5 ± 5.9 (13) 305.8 ± 3.7 (13)t
Birth Wt. (g)
Liveborn
5.4 + 0.2 (14) 5.7 + 0.2 (13) 6.2 ± 1.0 (13)§
12.9 _+ 1.2 14.0 +_ 0.8 13.4 ± 1.3
Males 47.8 + 1.0 48.0 ± 1.2 51.6 ± 1.3 II
Females 46.2 ± 0.8 46.1 ± 0.9 50.5 + 1.1 II
E, ethanol; PF, pair-fed; C, control. ( ) = Number of litters; G21 = gestation day 21; L1 = lactation day 1 (within 24 h of birth). Values represent mean + SEM. *Weight gain over gestation, Group x days F(6, 108) = 8.84,p < 0.001; from days 7-21, C > E = PF, p's < 0.01. tGroupeffect, F(2, 36) = 4.12, p < 0.05; E > PF = C , p < 0.05. ~Group effect, F(2, 37) = 6.51,p < 0.01; C > E = PF, p < 0.05. §Groupeffect, F(2, 37) = 5.38,p < 0.01; C > E = PF, p < 0.05. PIGroupeffect, F(2, 74) = 9.34, p < 0.001; C > E = PF, p < 0.01.
G e s t a t i o n length was increased slightly but significantly in E females c o m p a r e d with b o t h P F a n d C females (p's < 0.05). G r o u p s did n o t differ in n u m b e r o f live-born young. H o w e v e r , b i r t h weights o f b o t h E a n d P F o f f s p r i n g were less t h a n weights o f C o f f s p r i n g (p's < 0.05), a n d these differences persisted t h r o u g h weaning o n day 22 o f age (p's < 0.05) (Table 1). In a d d i t i o n , there was a t r e n d ( p = 0.08) t o w a r d a n effect o f sex; overall, males pups weighed slightly m o r e t h a n female pups at weaning.
Adrenocortical Response to Restraint Stress Due to differences in time points m e a s u r e d d u r i n g restraint stress, as well as n o r m a l sex differences in p l a s m a corticoster o n e levels (11), the time course o f a d r e n o c o r t i c a l response to restraint was analyzed separately for males a n d females. F o r females, the A N O V A revealed n o significant effect o f g r o u p a n d n o g r o u p x time interaction. T h e only significant effect
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was t h a t for time ( p < 0.001) (Fig. 1). T h e r e was a m a r k e d increase in p l a s m a corticosterone over basal levels at 30-min restraint in all females ( p < 0.01), a n d corticoid levels rem a i n e d significantly elevated t h r o u g h 150-rain restraint. By 180 min, there was a significant decrease f r o m peak levels t o w a r d basal levels ( p < 0.01); however, corticosterone rem a i n e d significantly elevated over basal levels t h r o u g h o u t the 240-min stress period (p's < 0.01). In contrast, the A N O V A for males revealed significant effects o f b o t h g r o u p ( p < 0.05) a n d time ( p < 0.001) (Fig. 2). Overall, E males h a d higher p l a s m a corticosterone levels t h a n P F males ( p < 0,05); the difference between E a n d C males a p p r o a c h e d significance ( p < 0.10). F u r t h e r post hoc analyses indicated t h a t males in all g r o u p s showed a m a r k e d corticosterone increase over basal levels at 30-rain restraint ( p < 0.01), a n d corticoid levels r e m a i n e d significantly elev a t e d t h r o u g h 90-rain restraint ( p < 0.05). By 120 min, P F
40
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90 120 150 180 210 240 Time (minutes)
FIG. 1. Plasma corticosterone (mean + SEM) time course during restraint stress in females. E, ethanol; PF, pair-fed; C, control. Time 0 = basal levels; n's per group = 6-7. For stress time points, n's per group = 7-9. Time effect, F(2, 165) = 72.6, p < 0.001 (see text for full discussion).
.
0
30
60
90
120 150 180 210 240
Time (minutes) FIG. 2. Plasma corticosterone (mean + SEM) time course during restraint stress in males. E, ethanol; PF, pair-fed; C, control. Time 0 = basal levels; n per group = 7. For stress time points, n's per group = 7-10. Group effect, F(2, 147) = 3.5, p < 0.05; E > PF, p < 0.05; E > C, p < 0.10. Time effect, F(6, 147) = 42.2, p < 0.001 (see text for full discussion).
222
WEINBERG
and C males showed a significant corticoid decrease from peak levels toward basal levels, (p's < 0.05), although corticosterone remained significantly elevated over basal levels throughout the 240-min restraint period (p's < 0.05). E males, however, showed no significant decrease from peak corticosterone levels throughout the 240-rain stress period (all time points greater than basal, p's < 0.05); at 180 min, group differences also reached significance (E > PF = C, p < 0.01). DISCUSSION
Previous studies suggested that prenatal ethanol exposure results in pituitary-adrenal hyperresponsiveness to stressors primarily in fetal ethanol-exposed females but not males. Consistent evidence of enhanced pituitary-adrenal activation and/ or delayed recovery back to basal levels in E compared with PF and C females has been reported, whereas E males have typically been found to be similar in response to males in control conditions (10,14,23,24,30,32). Results of the present study indicate that H P A hyperresponsiveness is not limited to fetal ethanol-exposed females, but may be demonstrated in fetal ethanol-exposed males under appropriate conditions. During prolonged restraint stress, E males were not different from PF and C males in their initial response to restraint, but showed a greater and more prolonged corticoid elevation over the 4-h stress period. Under these same test conditions, however, E females did not differ from PF and C females in their adrenocortical response to stress. These data differ from those reported in our previous study which examined a l-h period of restraint stress (30). In that study, corticosterone levels of males from E, PF, and C conditions did not differ from each other, and all males showed elevated corticoids at 30 and 60 min of restraint. In contrast, corticosterone levels of PF and C females appeared to peak at 30 rain and then decrease significantly by 60 rain of restraint, whereas E females showed no significant corticoid decrease from 30 to 60 rain. One possible reason for the differences in results between these two studies is that the restraint conditions were different. That is, in the present study, the restraint tubes, although slightly longer than those used previously, were smaller in diameter, and therefore, animals were more tightly restrained. For females, this increase in the severity or intensity of the restraint stress resulted in a more sustained peak elevation of corticosterone (peak levels were maintained for 90 min or longer in the present study as compared with 30 min in the previous study) and possibly, a maximal response in females from all groups, thus masking or eliminating the differential responsiveness seen previously. For males, on the other hand, the peak corticoid levels observed at 30 and 60 min in the present study were similar to those observed in the previous study. However, the longer time course of the present study revealed a differential responsiveness among groups (i.e., E males showed a more sustained corticoid elevation than PF and C males) that could not be observed with the 1-h restraint period used previously. Thus, it appears that H P A hyperresponsiveness may occur in both males and females following in utero ethanol exposure. Demonstration of differential stress responsiveness in E males and females appears to depend on the parameters of the test situation, including the nature and intensity of the stressor and the time course of the response during stress or recovery from stress that is measured. Previous studies by Taylor and coworkers suggest that enhanced pituitary-adrenal responsiveness to stress in fetal
ethanol-exposed offspring is not due to increased responsiveness of either the adrenal or the pituitary to its secretagogues. Adrenal sensitivity to adrenocorticotropic hormone (ACTH) was examined in dexamethasone-suppressed rats (24). Plasma corticosterone levels were found to be similarly suppressed by dexamethasone in E, PF, and C animals, and ACTH was found to produce similar dose-dependent elevations in plasma corticosterone in all three groups. Pituitary sensitivity to corticotropin releasing factor (CRF), arginine vasopressin (AVP), or vehicle was examined in rats pretreated with chlorpromazinc, morphine, and pentobarbital to suppress endogenous release of A C T H secretagogues (26). It was found that all rats responded to AVP and CRF with marked increases in ACTH and corticosterone; responses of E rats were not greater than those of PF and C rats. Together, these data suggest that a defect in regulation of H P A function probably lies within the CNS (26). In a recent study (33) we tested the hypothesis that H P A hyperresponsiveness in fetal ethanol-exposed offspring may be due to a deficit in feedback control of pituitary-adrenal activity. It is well-established that the glucocorticoids exert a negative feedback action on the pituitary, hypothalamus, and other brain areas to regulate production and secretion of hormones of the H P A axis (9). Glucocorticoid-binding sites have been demonstrated in the pituitary, the hypothalamus, and limbic system structures, particularly the hippocampus, amygdala, and septum (9,12). Recent studies suggest a major role for the hippocampus in the negative feedback actions of the glucocorticoids (5,12,15-17). Importantly, the hippocampus is also one of the most vulnerable areas within the brain to the teratogenic effects of prenatal ethanol exposure (3,34). Thus, we tested the hypothesis that a long-term decrease in hippocampal glucocorticoid receptor concentration might underlie this proposed deficit in feedback regulation in fetal ethanol-exposed animals. We found no significant differences in maximal binding or binding affinity of either type I or type II glucocorticoid receptors among animals from E, PF, and C groups tested under basal (i.e., nonstressed) conditions. Consistent with previous studies (28), however, females showed significantly greater maximal binding and significantly lower binding affinity than males; prenatal ethanol exposure did not alter this sex difference. Although these data do not support our original hypothesis, they do not rule out the possibility that an ethanol-induced deficit in feedback control of H P A activity does in fact exist. Some support for this suggestion comes from data of Taylor and coworkers who reported an altered temporal profile of either suppression or sensitivity to dexamethasone (13), as well as a slower decline of ACTH to basal levels following footshock stress (27) in E as compared with PF and C rats. It is possible that feedback regulation of pituitary-adrenal activity may be intact at the level of the hippocampus but deficient at the hypotbalamic or pituitary level. Alternatively, differential regulation of glucocorticoid receptors may occur in E compared with PF and C animals during stress. For example, data indicate that in utero ethanol exposure does not alter resting levels of corticosterone (22,30) nor the normal development of circadian rhythmicity of basal pituitary-adrenal function (22), but rather, affects only stress and drug responsiveness of the offspring H P A axis (14, 23,24,30,32). Thus, it is possible that, compared with PF and C animals, E animals may exhibit changes in receptor occupancy and translocation to the nucleus during acute stress, or increased down-regulation a n d / o r delayed recovery of recep-
P R E N A T A L S T R E S S A N D STRESS R E S P O N S I V E N E S S
223
tor concentration during or following chronic stress. These possibilities remain to be investigated. Finally, these data may have important clinical implications. Children prenatally exposed to alcohol are known to be hyperactive, uninhibited, and impulsive in behavior, and to have attentional deficits that may reflect an inability to inhibit responses (18-21). These behavior deficits are particularly noticeable in challenging or stressful situations (20). Our data suggest that deficits in pituitary-adrenal response inhibition or recovery following stress could accompany or perhaps even exacerbate these behavioral deficits, as A C T H , C R F , and the glucocorticoids are known to have m o d u l a t o r y effects on be-
havioral responses to stressful situations (12). Such central effects o f these hormones would thus further compromise the child's ability to respond appropriately to challenging or stressful situations. ACKNOWLEDGEMENTS The expert assistance of Gretta D'Alquen, Edna Halabe, and Catherine Bachewich is gratefully acknowledged. The author thanks Marian O'Connor and Patricia Jeffs for preparation of the manuscript. A portion of these data was presented at the Research Society on Alcoholism Meeting, Beaver Creek, CO, June 1989. This work was supported by National Institute on Alcohol Abuse and Alcoholism, Grant AA07789.
REFERENCES 1. Abel, E. L.; Jacobson, S.; Sherwin, B. T. In utero alcohol exposure: Functional and structural brain damage. Neurobehav. Toxicol. Teratol. 5:363-366; 1983. 2. Angelogianni, P.; Gianoulakis, C. Prenatal exposure to ethanol alters the ontogeny of the B-endorphin response to stress. Alcohol. Clin. Exp. Res. 13:564-571; 1989. 3. Barnes, D. E.; Walker, D. W. Prenatal ethanol exposure permanently reduces the number of pyramidal neurons in rat hippocampus. Dev. Brain Res. 1:333-340; 1981. 4. Davidson, J. M.; Jones, L. E.; Levine, S. Feedback regulation of adrenocorticotropin secretion in "basal" and "stress" conditions: Acute and chronic effects of intrahypthalamic corticoid implantation. Endocrinology 82:655-663; 1968. 5. DeKloet, E. R.; Reul, J. M. H. M. Feedback action and toxic influence of corticosteroids on brain function: A concept arising from the heterogeneity of brain receptor systems. Psychoneuroendocrinology 12:83-105; 1987. 6. Gallo, P. V.; Weinberg, J. Corticosterone rhythmicity in the rat: Interactive effects of dietary restriction and schedule of feeding. J. Nutr. 111:208-218; 1981. 7. Kakihana, R.; Butte, J. C.; Moore, J. A. Endocrine effects of maternal alcoholization: Plasma and brain testosterone, dehydrotestosterone, estradiol and corticosterone. Alcohol. Clin. Exp. Res. 4:57-61; 1980. 8. Kaneko, M.; Kaneko, K.; Shinsako, J.; Dallman, M. F. Adrenal sensitivity to adrenocorticostropin varies diurnally. Endocrinology 109:70-75; 1981. 9. Keller-Wood, M. E.; Dallman, M. F. Corticosteroid inhibition of ACTH secretion. Endocrinol. Rev. 5:1-24; 1984. 10. Kelley, S. J.; Mahoney, J. C.; Randich, A.; West, J. R. Indices of stress in rats: Effects of sex, perinatal alcohol and artificial rearing. Physiol. Behav. 49:751-765; 1991. 11. Kitay, J. T. Sex differences in adrenal cortical secretion in the rat. Endocrinology 68:818-824; 1961. 12. McEwen, B. S.; DeKloet, E. R.; Rostene, W. Adrenal steroid receptors and actions in the nervous system. Physiol. Rev. 66: 1121-1188; 1986. 13. Nelson, L. R.; Redei, E.; Liebeskind, J. C.; Branch, B. J.; Taylor, A. N. Corticosterone response to dexamethasone in fetal ethanol-exposed rats. Proc. West. Pharmaeol. Soc. 28:299-302; 1985. 14. Nelson, L. R.; Taylor, A. N.; Lewis, J. W.; Poland, R. E.; Redei, E.; Branch, B. J. Pituitary-adrenal responses to morphine and footshock stress are enhanced following prenatal alcohol exposure. Alcohol. Clin. Exp. Res. 10:397-402; 1986. 15. Reul, J. M. H. M.; DeKloet, E. R. Two receptor systems for corticosterone in rat brain: Microdistribution and differential occupation. Endocrinology 117:2505-2511; 1985. 16. Sapolsky, R. M.; Krey, L. C.; McEwen, B. S. Glucocorticoidsensitive hippocampal neurons are involved in terminating the adrenocortical stress response. Proc. Nat. Acad. Sci. 81:61746177; 1984. 17. Sapolsky, R. M.; Krey, L. C.; McEwen, B. S. The neuroendocrinology of stress and aging: The glucocorticoid cascade hypothesis. Endocrinol. Rev. 7:284-301; 1986.
18. Shaywitz, B. A.; Griffieth, G. G.; Warshaw, J. B. Hyperactivity and cognitive deficits in developing rat pups born to alcoholic mothers: An experimental model of the expanded fetal alcohol syndrome (EFAS). Neurobehav. Toxicol. 1:113-122; 1979. 19. Streissguth, A. P.; Barr, H. M.; Martin, D. C. Maternal alcohol use and neonatal habituation assessed with the Brazelton Scale. Child Dev. 54:1109-1118; 1983. 20. Streissguth, A. P.; Barr, H. M.; Sampson, P. D.; ParrishJohnson, J. C.; Kirchner, G. L.; Martin, D. C. Attention, distraction and reaction time at age 7 years and prenatal alcohol exposure. Neurobehav. Toxicol. Teratol. 8:717-725; 1986. 21. Streissguth, A. P.; Clarren, S. K.; Jones, K. L. Natural history of the fetal alcohol syndrome: A 10-year follow-up of eleven patients. Lancet 10:85-92; 1985. 22. Taylor, A. N.; Branch, B. J.; Cooley-Matthews, B.; Poland, R. E. Effects of maternal ethanol consumption on basal and rhythmic pituitary-adrenal function in neonatal offspring. Psychoneuroendocrinology 7:49-58; 1982. 23. Taylor, A. N.; Branch, B. J.; Liu, S.; Weichmann, A. F.; Hill, M. A.; Kokka, N. Fetal exposure to ethanol enhances pituitaryadrenal and temperature responses to ethanol in adult rats. Alcohol. Clin. Exp. Res. 5:237-246; 1981. 24. Taylor, A. N.; Branch, B. J.; Liu, S. H.; Kokka, N. Long-term effects of fetal ethanol exposure on pituitary-adrenal responses to stress. Pharmacol. Biochem. Behav. 16:585-589; 1982. 25. Taylor, A. N.; Branch, B. J.; Nelson, L. R.; Lane, L. A.; Poland, R. E. Prenatal ethanol and ontogeny of pituitary-adrenal responses to ethanol and morphine. Alcohol 3:255-259; 1986. 26. Taylor, A. N.; Branch, B. J.; Van Zuylen, J. E.; Redei, E. Maternal alcohol consumption and stress responsiveness in offspring. In: Chrousos, G. P.; Loriaux, D. L.; Gold, P. W.; eds. Advances in experimental medicine and biology, vol. 245. Mechanisms of physical and emotional stress. New York: Plenum Press; 1988: 311-317. 27. Taylor, A. N.; Branch, B. J.; Van Zuylen, J. E.; Redei, E. Prenatal ethanol exposure alters ACTH stress response in adult rats. Alcohol. Clin. Exp. Res. 10:120; 1986. 28. Turner, B. B.; Weaver, D. A. Sexual demorphism of glucocorticoid binding in rat brain. Brain Res. 343:16-23; 1985. 29. Weinberg, J. Effects of ethanol and maternal nutritional status on fetal development. Alcohol. Clin. Exp. Res. 9:49-55; 1985. 30. Weinberg, J. Hyperresponsiveness to stress: Differential effects of prenatal ethanol on males and females. Alcohol. Clin. Exp. Res. 12:647-652; 1988. 31. Weinberg, J. Prenatal ethanol exposure alters adrenocortical development of offspring. Alcohol. Clin. Exp. Res. 13:73-83; 1989. 32. Weinberg, J.; Gallo, P. V. Prenatal ethanol exposure: Pituitaryadrenal activity in pregnant dams and offspring. Neurobehav. Toxicoi. Teratol. 4:515-520; 1982. 33. Weinberg, J.; Petersen, T. D. Effects of prenatal ethanol exposure on glucocorticoid receptors in rat hippocampus. Alcohol. Clin. Exp. Res. 15:711-716; 1991. 34. West, J. R.; Pierce, D. R. Perinatal alcohol exposure and neuronal damage. In: West, J. R., ed. Alcohol and brain development. New York: Oxford University Press; 1986:120-157.
0741-8329/92$5.00 + .130 Copyright©1992PergamonPress Ltd.
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Prenatal Ethanol Effects: Sex Differences in Offspring Stress Responsiveness JOANNE W E I N B E R G
Department o f Anatomy, Faculty o f Medicine, The University o f British Columbia, Vancouver, BC V6T 1Z3 Canada Received 28 M a y 1991; Accepted 6 D e c e m b e r 1991 WEINBERG, J. Prenatal ethanol effects: Sex differences in offspring stress responsiveness. ALCOHOL 9(3) 219-223, 1992.-Previous studies have shown that offspring prenatally exposed to ethanol are hyperresponsive to stressors in adulthood, and have suggested that females are typically more affected than males. The present study was undertaken to investigate further this apparent sex difference in prenatal ethanol effects on stress responsiveness. Male and female offspring from prenatal ethanol-exposed (E), pair-fed (PF), and ad lib-fed control (C) conditions were tested in adulthood to determine adrenoconical responses to a prolonged (4-h) restraint stress. There were no significant differences in corticoid responsiveness among females from the three treatment groups. All females showed a marked increase in plasma corticosterone at 30 rain, and corticoid levels remained elevated through 150-min restraint. By 180 min, all females showed a significant corticoid decrease, although corticosterone remained elevated over basal levels throughout the 240-rain stress period. For males, in contrast, there were significant differences among groups. All males showed a significant corticoid increase over basal levels at 30 min, and corticoids remained significantly elevated through 90-min restraint. By 120 min, PF and C males showed a significant corticoid decrease although corticoids never returned to basal levels during the 240-min restraint period. E males, however, showed no significant decrease from peak corticosterone levels throughout the 240-min restraint stress. These data indicate that pituitary-adrenal hyperresponsiveness is not limited to fetal ethanol-exposed females, but may be demonstrated in fetal ethanol-exposed males under appropriate conditions. The parameters of the test situation, including the nature and intensity of the stressor and the time course of the response during stress or recovery from stress that is measured, appear to be critical in revealing differential effects of prenatal ethanol exposure on males and females. Fetal alcohol syndrome Sex differences Hyperresponsiveness to stress Rat
Stress
Pituitary-adrenal system
ETHANOL consumption by the pregnant female rat alters development and stress responsiveness of the offspring hypothalamo-pituitary-adrenal (HPA) axis. Fetal ethanolexposed (E) offspring have elevated brain and plasma corticosterone levels at birth (7,22,31), and show reduced adrenocortical responses to stressors and drug (e.g., ethanol, morphine) challenges during the preweaning period compared with PF and C offspring (25,31). /~-Endorphin (~-EP) responses to stressors are similarly suppressed in E compared with PF and C offspring prior to weaning (2). Interestingly, such reduced stress responsiveness is a transient phenomenon. Beginning in the period between weaning age and puberty, and persisting into adulthood, E animals often exhibit hyperresponsiveness to stressors compared with controls. Enhanced pituitaryadrenal activation to intermittent footshock, ether, psychological or neurogenic stressors, and drugs such as ethanol and morphine, as well as increased /~-EP responses to cold and ether stress have been reported in E compared with PF and C animals (2,14,23,24,27,30,32). In addition, E offspring may
also exhibit deficits in response inhibition or recovery of the pituitary-adrenal system toward basal levels following stress
(30). An important finding in these previous studies, at least with regard to the HPA axis, is that females appear to be more vulnerable than males to prenatal ethanol effects on stress responsiveness. Data from Taylor and coworkers (14, 23,24,27) demonstrating HPA hyperresponsiveness in E animals were derived primarily from females. Studies in our laboratory found deficits in adrenocortical response inhibition or recovery following stress only in E females; E males were similar in response to PF and C males (30). Similarly, a recent study by Kelley et al. (10) found that early postnatal ethanol exposure resulted in adrenocortical hyperresponsiveness in a forced swimming task in female but not male offspring. The present study was undertaken to investigate further this apparent sex difference in effects of prenatal ethanol exposure on stress responsiveness. Previous studies typically used stressors that were acute rather than chronic and/or of
Requests for reprints should be addressed to Dr. Joanne Weinberg, Department of Anatomy, The University of British Columbia, 2177 Wesbrook Mall, Vancouver, BC V6T IZ3, Canada. 219
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relatively short duration. In the present study, adrenocortical responses to a prolonged s t r e s s o r - 4 - h r e s t r a i n t - w e r e examined. METHOD
Animals and Mating Forty Sprague-Dawley female rats (250-275 g; Canadian Breeding Farms, St. Constant, Quebec) were group housed in polycarbonate cages (46 x 24 × 15 cm) for a 1-2-week adaptation period. They were then housed singly in stainlesssteel suspended cages with wire mesh front and floor (25 x 18 × 18 cm) together with a Sprague-Dawley male (300-350 g), and cage papers were checked daily. The presence of a vaginal plug indicated day 1 of gestation. During adaptation and mating all animals had ad lib access to standard laboratory chow (Ralston Purina of Canada, Woodstock, Ontario) and water. The colony room had controlled temperature (2122°C) and lighting, with lights on 0600-1800 h.
Diets and Feeding On day 1 of gestation, females were randomly assigned to one of three groups: (1) ethanol (E), n = 14: liquid ethanol diet, ad lib; (2) pair-fed (PF), n = 13: liquid control diet, with each animal pair-fed the amount consumed by a female in the ethanol group (g/kg body weight per day of gestation); (3) control (C), n = 13: laboratory chow and water, ad lib. Diets were prepared by Bio-Serv, Inc. (Frenchtown, N J), and were formulated to provide adequate nutrition to pregnant females regardless of ethanol intake (29). The liquid ethanol diet contained 36% ethanol-derived calories; maltose-dextrin was isocalorically substituted for ethanol in the liquid control diet. Fresh diet was placed on the cages daily in the late afternoon (1-2-h prior to lights off); bottles from the previous day were removed and weighed at this time to determine amount consumed. This feeding schedule was designed to prevent a shift in the corticosterone circadian rhythm which typically occurs in animals on a restricted feeding schedule such as that of the pair-fed group (6). Females were weighed on days 1, 7, 14, and 21 of gestation. On the afternoon of day 22, experimental diets were replaced with laboratory chow and water. At birth (designated day 1 lactation), cages were cleaned, dam and pups were weighed, and all litters were culled to ten (five males, five females when possible). For litters of eight or nine pups, pups from a dam of the same treatment condition, born on the same day, were fostered in to bring the litter size to ten. Litters with fewer than eight pups were eliminated from the study. Other than once weekly weighing and cage cleaning, dam and pups remained undisturbed until weaning on day 22, after which offspring were group housed by litter and by sex until testing in adulthood.
Testing and Blood Sampling Testing began when animals were 80-90 days of age. No more than one male and one female from any litter were assigned to any one blood sampling time, and each animal was blood sampled only once in this study. Animals were individually housed for at least 1 week prior to testing. All testing was conducted between 0800-1300 h. On the test day, animals were assigned either to the basal or to the stress condition. Cages were quickly and quietly carried from the colony room
to an adjacent laboratory. For animals in the basal condition, blood samples (0.5 cc) were obtained immediately by cardiac puncture under light ether anesthesia. Animals in the stress condition were quickly removed from their cages and immediately confined to plastic (PVC) restraint tubes which restricted movement. Tubes used for males were 6.5 cm in diameter x 20.5 cm in length; tubes for females were 5 cm in diameter × 15.5 cm in length. At intervals throughout the 4-h restraint period, animals were removed from their restraint tubes and blood samples obtained by cardiac puncture as described above. Samples from males were taken at 30, 60, 90, 120, 180, and 240 min of restraint; samples from females were taken at 30, 60, 90, 150, 180, 210, and 240 min of restraint. The entire sampling procedure was completed within 2 min of touching the animal's home cage (for basal samples) or restraint tube (for stress samples), which is rapid enough to obtain a reliable measure of corticosterone without any effects of disturbance or of the procedure of etherization and cardiac puncture (4). The study was carried out over an approximately 4-week period; males and females from E, PF, and C conditions were represented on every test day.
Radioimmunoassay Plasma was extracted in absolute ethanol (1:10 vol/vol) and corticosterone measured by radioimmunoassay using our adaptation of the method of Kaneko et al. (8). Antiserum was obtained from Immunocorp (Montreal) PQ; tracer, [1,2,6,73H] corticosterone, was obtained from DuPont, Canada (NEN Research Products, Mississauga, Ontario) and unlabeled corticosterone was obtained from Sigma Chemical Co. (St. Louis, MO). Dextran-coated charcoal was used to absorb and precipitate free steroid after incubation. Samples were counted in Formula 989 (NEN Research Products). Total corticosterone (bound plus free) was determined by this method.
Statistical Analyses Data were analyzed by appropriate analyses of variance (ANOVAs) for the factors of group and time. Time was treated as a repeated measure for analysis of the developmental data and as a nonrepeated measure for analysis of corticosterone levels. Significant main or interaction effects were further analyzed by Newman-Keuls paired comparisons. ANOVA results are presented in the tables and figure legends. RESULTS
Developmental Data Ethanol intake of pregnant females was consistently high throughout gestation, averaging 9.6 +_ 0.28, 12.0 ± 0.28, and 11.6 ± 0.20 g/kg body weight for weeks l, 2, and 3 of gestation, respectively. We have shown previously that ethanol intake at these levels results in blood alcohol levels of approximately 145-155 mg/dl (29). A repeated measures ANOVA on maternal weight gain during pregnancy revealed significant effects of group (p < 0.01) and days (p < 0.001), as well as a significant group × days interaction (p < 0.001). Post hoc tests indicated that from gestation day 7 onward, E and PF females weighed less than C females (p's < 0.01) (day 21 data shown in Table 1). Similarly, E a n d P F females weighed less than C females on the day of birth (within 24-h postpartum) (p's < 0.05) (Table 1).
PRENATAL
STRESS AND STRESS RESPONSIVENESS
221
TABLE I PREGNANCY, BIRTH, AND DEVELOPMENTAL DATA FROM DAMS AND PUPS IN ETHANOL, PAIR-FED, AND CONTROL CONDITIONS Maternal Females
Pups Weaning Wt. (g)
E PF C
G21 B. Wt. (g)
Gestation Length (days)
350.0 + 6.3 (14) 352.0 ± 7.4 (13) 389.2 + 7.5 (12)*
23.36 ± 0.10 (14)t 22.96 _+ 0.09 (13) 23.04 ± 0.13 (13)
LIB. Wt. (g) 284.1 ± 2.7 (14) 292.5 ± 5.9 (13) 305.8 ± 3.7 (13)t
Birth Wt. (g)
Liveborn
5.4 + 0.2 (14) 5.7 + 0.2 (13) 6.2 ± 1.0 (13)§
12.9 _+ 1.2 14.0 +_ 0.8 13.4 ± 1.3
Males 47.8 + 1.0 48.0 ± 1.2 51.6 ± 1.3 II
Females 46.2 ± 0.8 46.1 ± 0.9 50.5 + 1.1 II
E, ethanol; PF, pair-fed; C, control. ( ) = Number of litters; G21 = gestation day 21; L1 = lactation day 1 (within 24 h of birth). Values represent mean + SEM. *Weight gain over gestation, Group x days F(6, 108) = 8.84,p < 0.001; from days 7-21, C > E = PF, p's < 0.01. tGroupeffect, F(2, 36) = 4.12, p < 0.05; E > PF = C , p < 0.05. ~Group effect, F(2, 37) = 6.51,p < 0.01; C > E = PF, p < 0.05. §Groupeffect, F(2, 37) = 5.38,p < 0.01; C > E = PF, p < 0.05. PIGroupeffect, F(2, 74) = 9.34, p < 0.001; C > E = PF, p < 0.01.
G e s t a t i o n length was increased slightly but significantly in E females c o m p a r e d with b o t h P F a n d C females (p's < 0.05). G r o u p s did n o t differ in n u m b e r o f live-born young. H o w e v e r , b i r t h weights o f b o t h E a n d P F o f f s p r i n g were less t h a n weights o f C o f f s p r i n g (p's < 0.05), a n d these differences persisted t h r o u g h weaning o n day 22 o f age (p's < 0.05) (Table 1). In a d d i t i o n , there was a t r e n d ( p = 0.08) t o w a r d a n effect o f sex; overall, males pups weighed slightly m o r e t h a n female pups at weaning.
Adrenocortical Response to Restraint Stress Due to differences in time points m e a s u r e d d u r i n g restraint stress, as well as n o r m a l sex differences in p l a s m a corticoster o n e levels (11), the time course o f a d r e n o c o r t i c a l response to restraint was analyzed separately for males a n d females. F o r females, the A N O V A revealed n o significant effect o f g r o u p a n d n o g r o u p x time interaction. T h e only significant effect
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was t h a t for time ( p < 0.001) (Fig. 1). T h e r e was a m a r k e d increase in p l a s m a corticosterone over basal levels at 30-min restraint in all females ( p < 0.01), a n d corticoid levels rem a i n e d significantly elevated t h r o u g h 150-rain restraint. By 180 min, there was a significant decrease f r o m peak levels t o w a r d basal levels ( p < 0.01); however, corticosterone rem a i n e d significantly elevated over basal levels t h r o u g h o u t the 240-min stress period (p's < 0.01). In contrast, the A N O V A for males revealed significant effects o f b o t h g r o u p ( p < 0.05) a n d time ( p < 0.001) (Fig. 2). Overall, E males h a d higher p l a s m a corticosterone levels t h a n P F males ( p < 0,05); the difference between E a n d C males a p p r o a c h e d significance ( p < 0.10). F u r t h e r post hoc analyses indicated t h a t males in all g r o u p s showed a m a r k e d corticosterone increase over basal levels at 30-rain restraint ( p < 0.01), a n d corticoid levels r e m a i n e d significantly elev a t e d t h r o u g h 90-rain restraint ( p < 0.05). By 120 min, P F
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90 120 150 180 210 240 Time (minutes)
FIG. 1. Plasma corticosterone (mean + SEM) time course during restraint stress in females. E, ethanol; PF, pair-fed; C, control. Time 0 = basal levels; n's per group = 6-7. For stress time points, n's per group = 7-9. Time effect, F(2, 165) = 72.6, p < 0.001 (see text for full discussion).
.
0
30
60
90
120 150 180 210 240
Time (minutes) FIG. 2. Plasma corticosterone (mean + SEM) time course during restraint stress in males. E, ethanol; PF, pair-fed; C, control. Time 0 = basal levels; n per group = 7. For stress time points, n's per group = 7-10. Group effect, F(2, 147) = 3.5, p < 0.05; E > PF, p < 0.05; E > C, p < 0.10. Time effect, F(6, 147) = 42.2, p < 0.001 (see text for full discussion).
222
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and C males showed a significant corticoid decrease from peak levels toward basal levels, (p's < 0.05), although corticosterone remained significantly elevated over basal levels throughout the 240-min restraint period (p's < 0.05). E males, however, showed no significant decrease from peak corticosterone levels throughout the 240-rain stress period (all time points greater than basal, p's < 0.05); at 180 min, group differences also reached significance (E > PF = C, p < 0.01). DISCUSSION
Previous studies suggested that prenatal ethanol exposure results in pituitary-adrenal hyperresponsiveness to stressors primarily in fetal ethanol-exposed females but not males. Consistent evidence of enhanced pituitary-adrenal activation and/ or delayed recovery back to basal levels in E compared with PF and C females has been reported, whereas E males have typically been found to be similar in response to males in control conditions (10,14,23,24,30,32). Results of the present study indicate that H P A hyperresponsiveness is not limited to fetal ethanol-exposed females, but may be demonstrated in fetal ethanol-exposed males under appropriate conditions. During prolonged restraint stress, E males were not different from PF and C males in their initial response to restraint, but showed a greater and more prolonged corticoid elevation over the 4-h stress period. Under these same test conditions, however, E females did not differ from PF and C females in their adrenocortical response to stress. These data differ from those reported in our previous study which examined a l-h period of restraint stress (30). In that study, corticosterone levels of males from E, PF, and C conditions did not differ from each other, and all males showed elevated corticoids at 30 and 60 min of restraint. In contrast, corticosterone levels of PF and C females appeared to peak at 30 rain and then decrease significantly by 60 rain of restraint, whereas E females showed no significant corticoid decrease from 30 to 60 rain. One possible reason for the differences in results between these two studies is that the restraint conditions were different. That is, in the present study, the restraint tubes, although slightly longer than those used previously, were smaller in diameter, and therefore, animals were more tightly restrained. For females, this increase in the severity or intensity of the restraint stress resulted in a more sustained peak elevation of corticosterone (peak levels were maintained for 90 min or longer in the present study as compared with 30 min in the previous study) and possibly, a maximal response in females from all groups, thus masking or eliminating the differential responsiveness seen previously. For males, on the other hand, the peak corticoid levels observed at 30 and 60 min in the present study were similar to those observed in the previous study. However, the longer time course of the present study revealed a differential responsiveness among groups (i.e., E males showed a more sustained corticoid elevation than PF and C males) that could not be observed with the 1-h restraint period used previously. Thus, it appears that H P A hyperresponsiveness may occur in both males and females following in utero ethanol exposure. Demonstration of differential stress responsiveness in E males and females appears to depend on the parameters of the test situation, including the nature and intensity of the stressor and the time course of the response during stress or recovery from stress that is measured. Previous studies by Taylor and coworkers suggest that enhanced pituitary-adrenal responsiveness to stress in fetal
ethanol-exposed offspring is not due to increased responsiveness of either the adrenal or the pituitary to its secretagogues. Adrenal sensitivity to adrenocorticotropic hormone (ACTH) was examined in dexamethasone-suppressed rats (24). Plasma corticosterone levels were found to be similarly suppressed by dexamethasone in E, PF, and C animals, and ACTH was found to produce similar dose-dependent elevations in plasma corticosterone in all three groups. Pituitary sensitivity to corticotropin releasing factor (CRF), arginine vasopressin (AVP), or vehicle was examined in rats pretreated with chlorpromazinc, morphine, and pentobarbital to suppress endogenous release of A C T H secretagogues (26). It was found that all rats responded to AVP and CRF with marked increases in ACTH and corticosterone; responses of E rats were not greater than those of PF and C rats. Together, these data suggest that a defect in regulation of H P A function probably lies within the CNS (26). In a recent study (33) we tested the hypothesis that H P A hyperresponsiveness in fetal ethanol-exposed offspring may be due to a deficit in feedback control of pituitary-adrenal activity. It is well-established that the glucocorticoids exert a negative feedback action on the pituitary, hypothalamus, and other brain areas to regulate production and secretion of hormones of the H P A axis (9). Glucocorticoid-binding sites have been demonstrated in the pituitary, the hypothalamus, and limbic system structures, particularly the hippocampus, amygdala, and septum (9,12). Recent studies suggest a major role for the hippocampus in the negative feedback actions of the glucocorticoids (5,12,15-17). Importantly, the hippocampus is also one of the most vulnerable areas within the brain to the teratogenic effects of prenatal ethanol exposure (3,34). Thus, we tested the hypothesis that a long-term decrease in hippocampal glucocorticoid receptor concentration might underlie this proposed deficit in feedback regulation in fetal ethanol-exposed animals. We found no significant differences in maximal binding or binding affinity of either type I or type II glucocorticoid receptors among animals from E, PF, and C groups tested under basal (i.e., nonstressed) conditions. Consistent with previous studies (28), however, females showed significantly greater maximal binding and significantly lower binding affinity than males; prenatal ethanol exposure did not alter this sex difference. Although these data do not support our original hypothesis, they do not rule out the possibility that an ethanol-induced deficit in feedback control of H P A activity does in fact exist. Some support for this suggestion comes from data of Taylor and coworkers who reported an altered temporal profile of either suppression or sensitivity to dexamethasone (13), as well as a slower decline of ACTH to basal levels following footshock stress (27) in E as compared with PF and C rats. It is possible that feedback regulation of pituitary-adrenal activity may be intact at the level of the hippocampus but deficient at the hypotbalamic or pituitary level. Alternatively, differential regulation of glucocorticoid receptors may occur in E compared with PF and C animals during stress. For example, data indicate that in utero ethanol exposure does not alter resting levels of corticosterone (22,30) nor the normal development of circadian rhythmicity of basal pituitary-adrenal function (22), but rather, affects only stress and drug responsiveness of the offspring H P A axis (14, 23,24,30,32). Thus, it is possible that, compared with PF and C animals, E animals may exhibit changes in receptor occupancy and translocation to the nucleus during acute stress, or increased down-regulation a n d / o r delayed recovery of recep-
P R E N A T A L S T R E S S A N D STRESS R E S P O N S I V E N E S S
223
tor concentration during or following chronic stress. These possibilities remain to be investigated. Finally, these data may have important clinical implications. Children prenatally exposed to alcohol are known to be hyperactive, uninhibited, and impulsive in behavior, and to have attentional deficits that may reflect an inability to inhibit responses (18-21). These behavior deficits are particularly noticeable in challenging or stressful situations (20). Our data suggest that deficits in pituitary-adrenal response inhibition or recovery following stress could accompany or perhaps even exacerbate these behavioral deficits, as A C T H , C R F , and the glucocorticoids are known to have m o d u l a t o r y effects on be-
havioral responses to stressful situations (12). Such central effects o f these hormones would thus further compromise the child's ability to respond appropriately to challenging or stressful situations. ACKNOWLEDGEMENTS The expert assistance of Gretta D'Alquen, Edna Halabe, and Catherine Bachewich is gratefully acknowledged. The author thanks Marian O'Connor and Patricia Jeffs for preparation of the manuscript. A portion of these data was presented at the Research Society on Alcoholism Meeting, Beaver Creek, CO, June 1989. This work was supported by National Institute on Alcohol Abuse and Alcoholism, Grant AA07789.
REFERENCES 1. Abel, E. L.; Jacobson, S.; Sherwin, B. T. In utero alcohol exposure: Functional and structural brain damage. Neurobehav. Toxicol. Teratol. 5:363-366; 1983. 2. Angelogianni, P.; Gianoulakis, C. Prenatal exposure to ethanol alters the ontogeny of the B-endorphin response to stress. Alcohol. Clin. Exp. Res. 13:564-571; 1989. 3. Barnes, D. E.; Walker, D. W. Prenatal ethanol exposure permanently reduces the number of pyramidal neurons in rat hippocampus. Dev. Brain Res. 1:333-340; 1981. 4. Davidson, J. M.; Jones, L. E.; Levine, S. Feedback regulation of adrenocorticotropin secretion in "basal" and "stress" conditions: Acute and chronic effects of intrahypthalamic corticoid implantation. Endocrinology 82:655-663; 1968. 5. DeKloet, E. R.; Reul, J. M. H. M. Feedback action and toxic influence of corticosteroids on brain function: A concept arising from the heterogeneity of brain receptor systems. Psychoneuroendocrinology 12:83-105; 1987. 6. Gallo, P. V.; Weinberg, J. Corticosterone rhythmicity in the rat: Interactive effects of dietary restriction and schedule of feeding. J. Nutr. 111:208-218; 1981. 7. Kakihana, R.; Butte, J. C.; Moore, J. A. Endocrine effects of maternal alcoholization: Plasma and brain testosterone, dehydrotestosterone, estradiol and corticosterone. Alcohol. Clin. Exp. Res. 4:57-61; 1980. 8. Kaneko, M.; Kaneko, K.; Shinsako, J.; Dallman, M. F. Adrenal sensitivity to adrenocorticostropin varies diurnally. Endocrinology 109:70-75; 1981. 9. Keller-Wood, M. E.; Dallman, M. F. Corticosteroid inhibition of ACTH secretion. Endocrinol. Rev. 5:1-24; 1984. 10. Kelley, S. J.; Mahoney, J. C.; Randich, A.; West, J. R. Indices of stress in rats: Effects of sex, perinatal alcohol and artificial rearing. Physiol. Behav. 49:751-765; 1991. 11. Kitay, J. T. Sex differences in adrenal cortical secretion in the rat. Endocrinology 68:818-824; 1961. 12. McEwen, B. S.; DeKloet, E. R.; Rostene, W. Adrenal steroid receptors and actions in the nervous system. Physiol. Rev. 66: 1121-1188; 1986. 13. Nelson, L. R.; Redei, E.; Liebeskind, J. C.; Branch, B. J.; Taylor, A. N. Corticosterone response to dexamethasone in fetal ethanol-exposed rats. Proc. West. Pharmaeol. Soc. 28:299-302; 1985. 14. Nelson, L. R.; Taylor, A. N.; Lewis, J. W.; Poland, R. E.; Redei, E.; Branch, B. J. Pituitary-adrenal responses to morphine and footshock stress are enhanced following prenatal alcohol exposure. Alcohol. Clin. Exp. Res. 10:397-402; 1986. 15. Reul, J. M. H. M.; DeKloet, E. R. Two receptor systems for corticosterone in rat brain: Microdistribution and differential occupation. Endocrinology 117:2505-2511; 1985. 16. Sapolsky, R. M.; Krey, L. C.; McEwen, B. S. Glucocorticoidsensitive hippocampal neurons are involved in terminating the adrenocortical stress response. Proc. Nat. Acad. Sci. 81:61746177; 1984. 17. Sapolsky, R. M.; Krey, L. C.; McEwen, B. S. The neuroendocrinology of stress and aging: The glucocorticoid cascade hypothesis. Endocrinol. Rev. 7:284-301; 1986.
18. Shaywitz, B. A.; Griffieth, G. G.; Warshaw, J. B. Hyperactivity and cognitive deficits in developing rat pups born to alcoholic mothers: An experimental model of the expanded fetal alcohol syndrome (EFAS). Neurobehav. Toxicol. 1:113-122; 1979. 19. Streissguth, A. P.; Barr, H. M.; Martin, D. C. Maternal alcohol use and neonatal habituation assessed with the Brazelton Scale. Child Dev. 54:1109-1118; 1983. 20. Streissguth, A. P.; Barr, H. M.; Sampson, P. D.; ParrishJohnson, J. C.; Kirchner, G. L.; Martin, D. C. Attention, distraction and reaction time at age 7 years and prenatal alcohol exposure. Neurobehav. Toxicol. Teratol. 8:717-725; 1986. 21. Streissguth, A. P.; Clarren, S. K.; Jones, K. L. Natural history of the fetal alcohol syndrome: A 10-year follow-up of eleven patients. Lancet 10:85-92; 1985. 22. Taylor, A. N.; Branch, B. J.; Cooley-Matthews, B.; Poland, R. E. Effects of maternal ethanol consumption on basal and rhythmic pituitary-adrenal function in neonatal offspring. Psychoneuroendocrinology 7:49-58; 1982. 23. Taylor, A. N.; Branch, B. J.; Liu, S.; Weichmann, A. F.; Hill, M. A.; Kokka, N. Fetal exposure to ethanol enhances pituitaryadrenal and temperature responses to ethanol in adult rats. Alcohol. Clin. Exp. Res. 5:237-246; 1981. 24. Taylor, A. N.; Branch, B. J.; Liu, S. H.; Kokka, N. Long-term effects of fetal ethanol exposure on pituitary-adrenal responses to stress. Pharmacol. Biochem. Behav. 16:585-589; 1982. 25. Taylor, A. N.; Branch, B. J.; Nelson, L. R.; Lane, L. A.; Poland, R. E. Prenatal ethanol and ontogeny of pituitary-adrenal responses to ethanol and morphine. Alcohol 3:255-259; 1986. 26. Taylor, A. N.; Branch, B. J.; Van Zuylen, J. E.; Redei, E. Maternal alcohol consumption and stress responsiveness in offspring. In: Chrousos, G. P.; Loriaux, D. L.; Gold, P. W.; eds. Advances in experimental medicine and biology, vol. 245. Mechanisms of physical and emotional stress. New York: Plenum Press; 1988: 311-317. 27. Taylor, A. N.; Branch, B. J.; Van Zuylen, J. E.; Redei, E. Prenatal ethanol exposure alters ACTH stress response in adult rats. Alcohol. Clin. Exp. Res. 10:120; 1986. 28. Turner, B. B.; Weaver, D. A. Sexual demorphism of glucocorticoid binding in rat brain. Brain Res. 343:16-23; 1985. 29. Weinberg, J. Effects of ethanol and maternal nutritional status on fetal development. Alcohol. Clin. Exp. Res. 9:49-55; 1985. 30. Weinberg, J. Hyperresponsiveness to stress: Differential effects of prenatal ethanol on males and females. Alcohol. Clin. Exp. Res. 12:647-652; 1988. 31. Weinberg, J. Prenatal ethanol exposure alters adrenocortical development of offspring. Alcohol. Clin. Exp. Res. 13:73-83; 1989. 32. Weinberg, J.; Gallo, P. V. Prenatal ethanol exposure: Pituitaryadrenal activity in pregnant dams and offspring. Neurobehav. Toxicoi. Teratol. 4:515-520; 1982. 33. Weinberg, J.; Petersen, T. D. Effects of prenatal ethanol exposure on glucocorticoid receptors in rat hippocampus. Alcohol. Clin. Exp. Res. 15:711-716; 1991. 34. West, J. R.; Pierce, D. R. Perinatal alcohol exposure and neuronal damage. In: West, J. R., ed. Alcohol and brain development. New York: Oxford University Press; 1986:120-157.
