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Effects of hyperactivity of the maternal hypothalamic-pituitary-adrenal (HPA) axis during pregnancy on the development of the HPA axis and brain monoamines of the offspring
ht. J. Devl Neuroscience, Vol. 12. No. 7, pp. 651-&Y, 1994
Pergaman
Etsevier Science Lrd Copyright 0 1994 ISDN Printed in Great Britain. All rights reserved 0736,5748/94 $7.cO+0.ca
EFFIZCTS OF H~ERA~IVITY OF THE MATERNAL HYPOT~LAMIC-PITUITARY-AD~NAL (HPA) AXIS DURING PREGNANCY ON THE DEVELOPMENT OF THE HPA AXIS AND BRAIN MONOAMINES OF THE OFFSPRING M. FAMELI,*
E. KITBAKI*
and F. ST~IANOPOUL~U~~
*Laboratory of Histology-Embryology, Athens University Medical School, Athens, Greece: TLaboratory of Biology-Biochemistry, Faculty of Nursing, IJniversity of Athens, P.O. Box 14224, Athens, 11510, Greece (Received 7 February 1994; revised 1 hcty 1994; accepted 6 July 1994)
Abstract-Offspring of mothers with adrenal hyperactivity during pregnancy have been reported to have changes in brain monoamines and altered emotional, reactive, sexual and maternal behavior. Since the hypothalamic-pituitary-adrenal (HPA) axis is known to be involved in the expression of such behaviors and is itself under monoaminergic control, we examined the development of the HPA axis and brain monoamines in pups whose mothers had adrenal hyperactivity, reflecting administration of ACTH during the last third of their pregnancy. The adrenals of the experimental animals weighed less and had aberrant mo~holo~. The abnormal histology was more pronounced in the adrenals of the experimental females than of the males, suggesting that females were more vulnerable to the prenatal treatment. In both experimental males and females, basal plasma corticosterone levels were higher compared to the controls, while after exposure to stress, experimental animals attained lower plasma corticosterone levels than the controls. In the brain of the experimental animals, dopaminergic activity appeared to be decreased, while serotonergic activity increased. Our results indicate that the prenatal treatment affected brain development in the offspring and as a consequence programmed the developing HPA axis in such a way as to hyperfunction under basal conditions, leading to its exhaustion and its inability to react properly to stress.
of the embryonic brain to high levels of circulating glucocorticoids is known to have a profound influence on its development. The consequences of such a treatment are often long-lasting20 and are expressed later in life, during adulthood, as aberrant behavior.6 Thus, offspring of mothers with adrenal hyperactivity during pregnancy have been reported to display and maternal deficits in learning abilityF3 in the propensity to playF6 in sexua17~8~L*,22,24 behavior,5v10 and to have altered emotionality25 and reactivity. 11J3 Since the HPA axis is known to be involved in the control of most of the above mentioned behaviors, we examined the development of the HPA axis in pups whose mothers had adrenal hyperactivity, as a result of ACTH injections, during the last third of their pregnancy. The function of the HPA axis was assessed when the offspring of both experimental and control mothers reached adulthood, by determining adrenal weights and histology, as well as the circulating levels of corticosterone under basal conditions and after exposure to stress. Brain type II glucocorticoid receptors were determined, since it is known that they are involved in the control of the stress response. 21 Furthermore, it has been shown that brain catecholaminergic16,27 and serotonergic16-18 activities are altered by prenatal stress and this has been proposed as the mechanism by which the effects of prenatal stress on adult behaviors are exerted. Based on the above, we determined the effect of our experimental treatment on brain monoamines and some of their metabolites. Exposure
EXPERIMENTAL
PROCEDURES
Animals
Long-Evans rats, reared in our laborato~, were kept under standard ~nditions (24”C, 12:12 hr light/dark cycle) and received food and water ad libitum. Vaginal smears were taken daily from breeder females and, when found in estrus, females were placed with males of the same strain. Pregnancy was determined the next morning by the presence of sperm in the vaginal smear $To whom all correspondence DN 12:7-C
should be addressed. 651
(=day 0 of pregnancy). C)n day 14 of gestation pregnant females were housed individually and randomly assigned to either the experimental or control group. When the offspring reached the age of 3-4 months. they were left undisturbed for 48 hr and blood was collected by cardiac puncture under ether anaesthesia from all animals, for the determination of basal plasma corticosterone levels. They were then left undisturbed for two weeks and at that time subjected to stress and blood collected again from all animals, for the determination of stress levels of corticosterone. Following this experiment. the animals were again left undisturbed for two weeks and then they were randomly assigned to two groups. Approximately half of the animals of each category (control males, control females. experimental males. experimental females) were used in the determination of brain type II gluc[~c~~rticoid receptors and the other half for the deterInination of brain monoanliIles. Animals used for the determination of brain glucocorticoid receptors were adrenalectomized one week prior to sacrifice. Adrenalectomies were performed via a dorsolateral approach under ether anesthesia. Adrenalectomized animals were maintained on 0.9% (w/v) NaCl as drinking solution. Animals were killed by intracardiac perfusion of 40 ml of ice-cold saline, under deep cthei anesthesia. Treatment
Pregnant females in the experimental group received daily S.C.injections of 0.08 ml of a suspension containing 8 IU of long-acting ACTH (Synachten Depot, Ciba; a zinc complex of a synthetic ACTH tetracosactide) during days 14-21 of pregnancy. Control mothers were injected with 0.08 ml of an aqueous suspension containing 10 mg benzyl alcohol per ml (the solvent of the ACTH suspensions, Adrenal weights and histology
The adrenals removed either at adrenalectomy, or at sacrifice, were weighed and then fixed in Bouin’s fluid. Both adrenals from each animal were weighed together, since no differences were found in weight between right and left adrenal. Adrenal histology was studied in S-Frn-thick paraffin sections stained with hematoxylin-eosin. Stress procedures
The animal mode1 of stress used consisted of 1 min of forced swimming in a 23°C water tank, followed by 15 min of confinement in a dark cylindrical box (18x9 cm). Animals were subjected to this stress at 1O:OOhr (4 hr into the light cycle) and blood samples for corticost~rone determination were collected immediately after. Measurement
0.f circulating corticnsterone
For the determination of basal levels of corticosterone, animals were left undisturbed for 48 hr prior to blood collection. Blood samples for both basal and stress levels of corticosterone were collected at 10~16hr by cardiac puncture under ether anesthesia. Plasma corticosterone levels were determined by RIA using a rat corticosterone antibody purchased from Sigma Chemical Co. This antiserum was generated against corticosterone-21 -thyroglobulin. It cross-reacted with 1 I -deoxycorticosterone (27%) and progesterone (17%). Assay sensitivity was 15 pg corticosterone per assay tube and coefficients of variation within and between assays were 12 and lo%, respectively (n=3). Unless otherwise stated, chemicals were purchased from Serva, Heidelberg, F.R.G. Determination
ofbrain
gfucmorticoid
receptors
Type II (classical glucocorticoid) receptors were determined in whole brain cytosol from adrenalectomized animals, by an in vitro competitive binding assay, using [3H]dexamethasone (Amersham; specific activity 42 Cifmmol) as the ligand, and the data were analysed by Scatchard analysis, as previously described.’ Five different Scatchard analyses were performed, each using brain cytosol from an individual rat. Every Scatchard analysis consisted of six assay points, each of which was done in triplicate. We determined type II brain glucocorticoid receptors in adrenalectomized animals, in order to avoid interference from endogenous glucocorticoids, whose levels are different between control and experimental animals, leading to differential occupation and thus masking of the receptors.
HPA development
in offspring of hyperactive HPA mothers
6.53
Determination of brain monoamines
Animals were killed by decapitation, their brains removed immediately and dissected on ice, as previously described. r Samples of brain areas were homogenized in 0.26 M perchloric acid containing 8 mM sodium metab~u~ite and 1.34 mM ethylene di~~etetraa~ti~ acid (EDTA). 5-Hydrox~~pta~ne (5-I-H), dopamine (DA), 5-hydroxy~dolea~tic acid (5HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (I-WA) were measured by high pressure liquid chromatography (HPLC) with an electrochemical detector (BAS LC-44) set at an electrochemical potential of 0.8 V. A Chrompack 28270 column was eluted with a mobile phase of 0.15 M phosphate buffer containing 16% methanol, 1 mM EDTA, 0.04 mM triethylamine, and 0.022% octanyl sulphate, at pH 3.9, using a flow rate of 0.3 mllrnin. Statistical analysis
Results were analysed by two-way analysis of variance (ANOVA), with subsequent t-testing. RESULT8 Adrenal weights
The adrenals of the male experimental animals weighed 150823.28 mg (n= 12) while the controls weighed 23.50-+3.81 mg (n=12). The equivalent values for the female animals were 263225.60 mg (n=12), compared to 32.0823.80 mg (n=12). These values represent the combined weight of both adrenals, since no statistically s~gnifi~nt differences were found between the weight of the left and right adrenal, in both control and experimental groups. Two-way ANOVA revealed statistically significant treatment [F(1,44)=35.63, P
Aberrant morphology was noted in the adrenals of the experimental animals, particularly the females. The zona fasciculata was not clearly delineated and its thickness was reduced; the normal fascicular arrangement of the cells was disorganized; a few cells of intensely eosinophilic and rarely “‘bubbly” cytoplasm could be seen, morphology suggestive of cellular degeneration; furthermore, there were areas containing only a few cells, the others seemingly having been lost and replaced by an amorphous eosinophilic substance (Fig. 1). No pronoun~d differences were seen in the zona reticularis between control and experimental animals. However, in the adrenals of the experimental group, the characteristic reticular organization was lost and instead cells were arranged homogeneously. In addition, the vesicular spaces could not be clearly seen. In the adrenal medulla, the only aberrant histological characteristic seen in the experimental and not the control adrenals was the presence of a few cells of lightly staining, almost clear cytoplasm. All the above-mentioned characteristics are suggestive of adrenal degeneration in the experimental group of animals. The abnormal histology was more pronounced in the adrenals of the experimental females, indicating that the females were more vulnerable to the prenatal exposure to high levels of circulating maternal glucocorticoids. Plasma corticosteru~e
HPA axis function was significantly altered in offspring of mothers that had received ACIH injections during pregnancy (Fig. 2). Two-way ANOVA showed statistically significant treatment and sex effects on both basal and stress-induced plasma levels of corticosterone: F(1,43)=15.43, P
hS4
M . Famcli ef ol.
respective effects of sex. On the other hand, no statistically significant treatment-sex interac Zion was found for either basal or stress-induced corticosterone levels. Subsequent paired i-te sting revealed that basal plasma corticosterone levels were higher in both male and female experimc entat animals, compared to the controls. In contrast, both male and female experimental animals attz lined lower corticosterone levels than the controls, after exposure to a stressful stimulus. Brain type II glucocorticoid receptors MOstatistically s~~~f~~nt differences were found either in the concentration fBmax>or the aff linity (Kd) of the gkcocorticoid receptor in the brain of the offspring as a result of the experimr Sntat treatment (Table I).
HPA development in offspring of hyperactive HPA mothers
655
Fig. 1. Effect of ACTH administration to the pregnant dam on the histology of the adrenals of the offspring when adult. Adrenals of control (la, b) and experimental (IIa, b) female animals. The bars delineate the limits of the fascicular zone (Ia, IIa): note the reduction in its thickness in Ila. Also note the disorganization of the normal fascicular arrangement of the ceils in the adrenals of the experimental animals (IIb). Arrow points to an area where cells seem to be missing (Iib). H & E. Ia and IIa X100; Ib and IIb X400.
Brain monoamines
Treatment of the pregnant dam with ACTH apparently affected the brain of the developing embryos in a permanent fashion, since differences in monoamines were found in the brains of the offspring when they reached adulthood (Tables 2 and 3). Two-way ANOVA showed a statistically si~ificant treatment effect on striatal DA [F(1,16)= 17.93, P=O.OOl] and DOPAC [F(l,16)=20.97, P=O.OOl] levels and a significant sex effect [F(1,16)=8,71, P=O.Oll], on cortical 5-HIAA levels. A significant treatment-sex interaction was found for striatal DOPAC [F(1,16)=5.49, P=O.O39] and 5-HIAA[F(1,16)=5.68,P=O.O36],as wellascortical5-HIAA [F(1,16)=8.44,P=O.O12]. Subsequent t-testing revealed that DA was decreased in the striatum of both experimental males and females, while DOPAC was decreased in the striatum of only the experimental males. On the other hand,
M. Fameli et al.
: e
1
I
HPA development
657
in offsp~ng of hyperactive HPA mothers
w
Control
c]
Experimental
I
BISll
After
Bad
After
ItWCP
strCss**
level**
strcss-
Fig. 2. Plasma levels of circulating corticosterone under basal conditions and after exposure to stress, in male and female control and experimental animals. Values represent means?S.D. Differences between experimental and control animals: *P
Bmax(fmoles per mg protein) Male Group Control Experimental
Female
Male
Female
Mean
S.D.
n
Mean
S.D.
n
Mean
S.D.
n
Mean
S.D.
n
474 395
117 104
5 5
409 380
117 98
5 5
3.35 3.49
1.53 1.72
5 5
3.62 3.47
1.82 1.75
5 5
5-HIAA was increased in both the striatum and the cerebral cortex of the experimental Other statistical significant differences were not found in brain monoamines.
maies.
DISCUSSION The hyperactivity of the maternal HPA axis during pregnancy (induced by injections of ACTH) profoundly affected the development of the HPA axis of the offspring. The active factor mediating the effects could be corticosterone, since it has been shown to circulate at high levels in the fetus, after exposure of the mother to stress.12,15 The consequences of the prenatal treatment were manifested at all levels of the HPA axis. In the brain, changes were found in the levels of dopamine, 3,4-dihydrox~henyla~tic acid and 5-hydroxyindoleacetic acid, suggesting that the prenatal treatment acted centrally to affect HPA function. Changes in serotonergic16-1s and catecholaminergi&27 activity as a result of prenatal stress have also been reported by others. Since the CNS of the experimental offspring was affected, one would expect altered levels of type II glucocorticoid receptors in the brain, since it is known that they play a key role in controlling the stress response. 21 Indeed it has been shown that there are fewer dexamethasone binding sites in the hippocampus of prenatally stressed female rats. 29 However, adrenalectomy abolished the difference29 and in our experiments we used cytosol from adrenalectomized animals for the determination of type II glucocorticoid receptors. This could explain the fact that we found no difference in dexamethasone binding sites in the brain between the experimental and control animals. The effects of the prenatal treatment on the brain could have acted in such a way as to program the basal level of adrenal function at a setting higher than the normal, resulting in high resting levels of circulating corticosterone, in spite of the histological appearance of the adrenal zona fasciculata, which showed signs of degeneration. It appears that the portion of adrenal tissue which remained intact, functioned more actively. Weinstock et al. 29 have also found higher resting levels of serum corticosterone in adult female rats exposed to a prenatal stress. The continuous hyperactivity of the
adrenal seems, however, to have led to its exhaustion. Adrenal weights were reduced and adrenat histology was suggestive of degeneration and the results were more pronounced in the females. A reduction of adrenal weight as a result of prenatal stress has also been reported by Suchecki and Paiermo Net~.~s The exhaustion of the adrenal cortex could be responsible for the inability to respond ade4uat~Iy to the ~halleu~~ of a stress. The experimentai animals thus had a defective emergency response, attaining lower corticosterone levels than the controls. Pollardi also found that offspring of stressed mothers had a defective emergency response. It is known that in humans, stress can precipitate depressive illness in predisposed individuals” and that elevated plasma giucocorticoids are commonly found in this iilness.‘4 Since antidepressants act by modif~ng serotonergi~ and dopami~~ergi~ activities, a defect in these neurotransmitter systems is considered to be one of the underIying bioIogicaI etiologicaf factors in depr~ss~o~.~~ Furthern~ore, females, both humans~ and rats.” are more vulnerable to the development of depression. Based on the above (with all due caution as to the relevance of animal models to the respective human situations), the experimental animals of our study can be considered to represent. an animal model of individuals predisposed to deveiop depression. Not only do they have high levels of ~ir~uIating glucocorfi~oids~ but they also tack an effective emergency response and are thus unable to adapt, since adaptation is mediated by the stress-induced rises of corticosferone3 Indeed it has been reported4 that. in rats, prenatal stress results in increased vulnerability and decreased habituation to stressful stimuli. On the other hand, the inability to successfully cope with or adapt to stress is known to be able to lead not only to altered affective states such as depression, but also to the deveIopment of ulcers, heart disease and hormonal imbalances.
REFERENCES 1. Alexis M. N., Styhanopoulou
F., Kjtraki E. and Sekeris K. ( 1983) The distribution and properties of the glucocorticoid receptor from rat brain and pituitary. J. BioI. C&em. 258,4?1&47t4. 2. Anisman ii. and Zacharko R. M. (1982) Depression: the predisposjng influence of stress. 5elznii 5&n Sti S. X%137. 3. Curson C. (1989) 5-Hydrox~ryptamine and corticosterone in an animal model of depression. Prq, ~el~ru-~~~c~~~~~~~r~ ma&. B&A ~~.~ck~ut.t3, X5-310. 4. Fride E.. Dan Y., Feldon J., Haievy G. and Weinstock N. (1986) Effects of prenatai stress on vulnerability to stress in prepubertal and adult rats. Pkysiol. Behav. 37,681-687. 'i . . Fride E., Dan Y.. Gavish M. and Weinstock M. (1985) Prenatal stress impairs maternal behavior in a conflict situation and reduces hippocampal benzodiazepine receptors. Life Sci. 36,2103-2109. 6. Grimm V. E. and Frieder B. (1987) The effects of mild maternal stress during pregnancy on the behavior of rat pups. IN. J. .~g~ri~~c~.3s,r%-72. 7‘ Herrenkohl L. R. (1979) Prenatal stress reduces f&&&y and fecundity in female offspring. Sc&cr 266,1~7-~~~~, 8. Herrenkoht L. R, and Whitney J. B. (1976) Effects of prepartat stress on postpartal nursing behavior, litter development and adult sexual behavior. Physiof. Bhav. 17,1019-1021. 0, Kennett G. A., Chaouloff F.. Marcou M. and Curzon G. (1986) Female rats are more vulnerable than males in an animal model of depression: the possible role of serotonin. Brain Res. 382,416-421. IO.Kin&y C. H. and Bridges R. S. (1988) Prenatal stress and maternal behavior in intact virgin rats: response latencies arc decreased in males and increased in females. i%~rm. Bekav. 22,76*9. 11.~~terpas~ua F., Chapman R. H. and Lore R. K. (1976) The effects of prenatal ps~cbo~o~ica~ stress on the sexual behavior and reactivity of male rats. Deal ~~~c~o~~~~~. 9,23S-245. t2.Montano M. M,, Wang M. H., Even M, D. and vom Saal F. S. (1991) Serum corticosterone in fetal mice: sex differences. circadian changes, and effect ofmaternalstress. Pkysiol. Bckav. 50,32.3-W). 13.Moore C. L. and Power K. L. (1986) Prenatal stress affects mother-infant interaction in Norway rats. Devl Prvchobir)/. 19,235-2&i. 14.Mu~by B. E. P. (1991) Steroids and depression, f. Sremi~l Biockem. M&c. SioL 38,537-559. IS.Dhkawa T.. Rohde W.. Takeshita S.. Domer G.. Arai K. and Okinawa S, f 1991) Effect of an acute maternal stress on the fetal bypothalamo-pituitary-adrenal system in late gestational Jig of the rat, E.xp. C&n. Endocrinol. 98, t23-129. lh.Peters D. A. V. (1982) Prenatal stress: effects on brain biogenic amine and plasma corticosterone levels. ~~~~~7~~~~~~. I
Biockem. Bekav. X7,721-725.
17.Peters D. A. V. (1986) Prenatal stress: effect on development of rat brain serotonergic neurons. Pharmacol. Riockem. Bekav. 24 1377-1382. 1x.Peters D. A. V. (1988) Both prenatal and postnatai factors contribute to the effects of maternal stress on offspring behavior and central S-hydrox~~ptamine receptors in the rat. ~~~rm~~o~. Biochent. Behar~.30,666673. 19.Pollard I. (1984) Effects of stress a~inister~d during pregancy on reprodu~~ve capacity and subsequent development of the offspring of rats: prolonged effects on litters of a second pregnancy. J. Endocritrot. t@O,301-306. 20. Pohard I. (1986) Prenatat stress effects over two generations in rats. J. ~~~cr~ffol. B&$239-244. 21.Reul J. M. II. MI, Van Den Bosch F. R. and De I&et E. R. (1987) Relative occupation of type-1 and type-11 corticosteroid receptors in rat brain following stress and dexamethasone treatment. J. Endacrinol. 115,459-467. 22. Rhees R. W. and Fleming D. E. (1981) Effects of malnutrition, maternal stress, or AC’TH injections during pregnancy on sexual behavior of male offspring. P~l~s~~. B&IV. 27,879-F-882.
HPA development
in offspring of hyperactive HPA mothers
23. Smith B. L., Wills G. and Naylor D. (1981) The effects of prenatal 24. 25. 26. 27. 28. 29. 30.
659
stress on rat offspring learning ability. J. Psychol. 107, 45-51. Stylianopoulou F. (1983) Effect of maternal alrenocorticotrophin injections on the differentiation of sexual behavior of the offspring. Horm. Behav. 17,324-331. Suchecki D. and Palermo Neto J. (1991) Prenatal stress and emotional response of adult offspring. Physiol.‘Behav. 49, 423426. Takahashi L. K., Haglin C. and Kalin N. H. (1992) Prenatal stress potentiates stress-induced behavior and reduces the propensity to play in juvenile rats. Physiol. Behav. 51,319323. Takahashi L. K., Turner J. G. and Kalin N. H. (1992) Prenatal stress alters brain catecholaminergic activity and potentiates stress-induced behavior in adult rats. Bruin Rex 574131-137. Van Praag H. M. (1982) Neurotransmitters and CNS disease: depression. Lmcet 1,1259-1264. Weinstock M., Matlina E., Maor G. I., Rosen H. and McEwen B. S. (1992) Prenatal stress selectively alters the reactivity of the hypothalamic-pituitary-adrenal system in the female rat. Brain Rex 595,195200. Weissman M. M. and Klerman G. (1977) Sex differences in the epidemiology of depression. Arch. Gen. Psychiaf. 34, 98111.
DN 12:7-D
Pergaman
Etsevier Science Lrd Copyright 0 1994 ISDN Printed in Great Britain. All rights reserved 0736,5748/94 $7.cO+0.ca
EFFIZCTS OF H~ERA~IVITY OF THE MATERNAL HYPOT~LAMIC-PITUITARY-AD~NAL (HPA) AXIS DURING PREGNANCY ON THE DEVELOPMENT OF THE HPA AXIS AND BRAIN MONOAMINES OF THE OFFSPRING M. FAMELI,*
E. KITBAKI*
and F. ST~IANOPOUL~U~~
*Laboratory of Histology-Embryology, Athens University Medical School, Athens, Greece: TLaboratory of Biology-Biochemistry, Faculty of Nursing, IJniversity of Athens, P.O. Box 14224, Athens, 11510, Greece (Received 7 February 1994; revised 1 hcty 1994; accepted 6 July 1994)
Abstract-Offspring of mothers with adrenal hyperactivity during pregnancy have been reported to have changes in brain monoamines and altered emotional, reactive, sexual and maternal behavior. Since the hypothalamic-pituitary-adrenal (HPA) axis is known to be involved in the expression of such behaviors and is itself under monoaminergic control, we examined the development of the HPA axis and brain monoamines in pups whose mothers had adrenal hyperactivity, reflecting administration of ACTH during the last third of their pregnancy. The adrenals of the experimental animals weighed less and had aberrant mo~holo~. The abnormal histology was more pronounced in the adrenals of the experimental females than of the males, suggesting that females were more vulnerable to the prenatal treatment. In both experimental males and females, basal plasma corticosterone levels were higher compared to the controls, while after exposure to stress, experimental animals attained lower plasma corticosterone levels than the controls. In the brain of the experimental animals, dopaminergic activity appeared to be decreased, while serotonergic activity increased. Our results indicate that the prenatal treatment affected brain development in the offspring and as a consequence programmed the developing HPA axis in such a way as to hyperfunction under basal conditions, leading to its exhaustion and its inability to react properly to stress.
of the embryonic brain to high levels of circulating glucocorticoids is known to have a profound influence on its development. The consequences of such a treatment are often long-lasting20 and are expressed later in life, during adulthood, as aberrant behavior.6 Thus, offspring of mothers with adrenal hyperactivity during pregnancy have been reported to display and maternal deficits in learning abilityF3 in the propensity to playF6 in sexua17~8~L*,22,24 behavior,5v10 and to have altered emotionality25 and reactivity. 11J3 Since the HPA axis is known to be involved in the control of most of the above mentioned behaviors, we examined the development of the HPA axis in pups whose mothers had adrenal hyperactivity, as a result of ACTH injections, during the last third of their pregnancy. The function of the HPA axis was assessed when the offspring of both experimental and control mothers reached adulthood, by determining adrenal weights and histology, as well as the circulating levels of corticosterone under basal conditions and after exposure to stress. Brain type II glucocorticoid receptors were determined, since it is known that they are involved in the control of the stress response. 21 Furthermore, it has been shown that brain catecholaminergic16,27 and serotonergic16-18 activities are altered by prenatal stress and this has been proposed as the mechanism by which the effects of prenatal stress on adult behaviors are exerted. Based on the above, we determined the effect of our experimental treatment on brain monoamines and some of their metabolites. Exposure
EXPERIMENTAL
PROCEDURES
Animals
Long-Evans rats, reared in our laborato~, were kept under standard ~nditions (24”C, 12:12 hr light/dark cycle) and received food and water ad libitum. Vaginal smears were taken daily from breeder females and, when found in estrus, females were placed with males of the same strain. Pregnancy was determined the next morning by the presence of sperm in the vaginal smear $To whom all correspondence DN 12:7-C
should be addressed. 651
(=day 0 of pregnancy). C)n day 14 of gestation pregnant females were housed individually and randomly assigned to either the experimental or control group. When the offspring reached the age of 3-4 months. they were left undisturbed for 48 hr and blood was collected by cardiac puncture under ether anaesthesia from all animals, for the determination of basal plasma corticosterone levels. They were then left undisturbed for two weeks and at that time subjected to stress and blood collected again from all animals, for the determination of stress levels of corticosterone. Following this experiment. the animals were again left undisturbed for two weeks and then they were randomly assigned to two groups. Approximately half of the animals of each category (control males, control females. experimental males. experimental females) were used in the determination of brain type II gluc[~c~~rticoid receptors and the other half for the deterInination of brain monoanliIles. Animals used for the determination of brain glucocorticoid receptors were adrenalectomized one week prior to sacrifice. Adrenalectomies were performed via a dorsolateral approach under ether anesthesia. Adrenalectomized animals were maintained on 0.9% (w/v) NaCl as drinking solution. Animals were killed by intracardiac perfusion of 40 ml of ice-cold saline, under deep cthei anesthesia. Treatment
Pregnant females in the experimental group received daily S.C.injections of 0.08 ml of a suspension containing 8 IU of long-acting ACTH (Synachten Depot, Ciba; a zinc complex of a synthetic ACTH tetracosactide) during days 14-21 of pregnancy. Control mothers were injected with 0.08 ml of an aqueous suspension containing 10 mg benzyl alcohol per ml (the solvent of the ACTH suspensions, Adrenal weights and histology
The adrenals removed either at adrenalectomy, or at sacrifice, were weighed and then fixed in Bouin’s fluid. Both adrenals from each animal were weighed together, since no differences were found in weight between right and left adrenal. Adrenal histology was studied in S-Frn-thick paraffin sections stained with hematoxylin-eosin. Stress procedures
The animal mode1 of stress used consisted of 1 min of forced swimming in a 23°C water tank, followed by 15 min of confinement in a dark cylindrical box (18x9 cm). Animals were subjected to this stress at 1O:OOhr (4 hr into the light cycle) and blood samples for corticost~rone determination were collected immediately after. Measurement
0.f circulating corticnsterone
For the determination of basal levels of corticosterone, animals were left undisturbed for 48 hr prior to blood collection. Blood samples for both basal and stress levels of corticosterone were collected at 10~16hr by cardiac puncture under ether anesthesia. Plasma corticosterone levels were determined by RIA using a rat corticosterone antibody purchased from Sigma Chemical Co. This antiserum was generated against corticosterone-21 -thyroglobulin. It cross-reacted with 1 I -deoxycorticosterone (27%) and progesterone (17%). Assay sensitivity was 15 pg corticosterone per assay tube and coefficients of variation within and between assays were 12 and lo%, respectively (n=3). Unless otherwise stated, chemicals were purchased from Serva, Heidelberg, F.R.G. Determination
ofbrain
gfucmorticoid
receptors
Type II (classical glucocorticoid) receptors were determined in whole brain cytosol from adrenalectomized animals, by an in vitro competitive binding assay, using [3H]dexamethasone (Amersham; specific activity 42 Cifmmol) as the ligand, and the data were analysed by Scatchard analysis, as previously described.’ Five different Scatchard analyses were performed, each using brain cytosol from an individual rat. Every Scatchard analysis consisted of six assay points, each of which was done in triplicate. We determined type II brain glucocorticoid receptors in adrenalectomized animals, in order to avoid interference from endogenous glucocorticoids, whose levels are different between control and experimental animals, leading to differential occupation and thus masking of the receptors.
HPA development
in offspring of hyperactive HPA mothers
6.53
Determination of brain monoamines
Animals were killed by decapitation, their brains removed immediately and dissected on ice, as previously described. r Samples of brain areas were homogenized in 0.26 M perchloric acid containing 8 mM sodium metab~u~ite and 1.34 mM ethylene di~~etetraa~ti~ acid (EDTA). 5-Hydrox~~pta~ne (5-I-H), dopamine (DA), 5-hydroxy~dolea~tic acid (5HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (I-WA) were measured by high pressure liquid chromatography (HPLC) with an electrochemical detector (BAS LC-44) set at an electrochemical potential of 0.8 V. A Chrompack 28270 column was eluted with a mobile phase of 0.15 M phosphate buffer containing 16% methanol, 1 mM EDTA, 0.04 mM triethylamine, and 0.022% octanyl sulphate, at pH 3.9, using a flow rate of 0.3 mllrnin. Statistical analysis
Results were analysed by two-way analysis of variance (ANOVA), with subsequent t-testing. RESULT8 Adrenal weights
The adrenals of the male experimental animals weighed 150823.28 mg (n= 12) while the controls weighed 23.50-+3.81 mg (n=12). The equivalent values for the female animals were 263225.60 mg (n=12), compared to 32.0823.80 mg (n=12). These values represent the combined weight of both adrenals, since no statistically s~gnifi~nt differences were found between the weight of the left and right adrenal, in both control and experimental groups. Two-way ANOVA revealed statistically significant treatment [F(1,44)=35.63, P
Aberrant morphology was noted in the adrenals of the experimental animals, particularly the females. The zona fasciculata was not clearly delineated and its thickness was reduced; the normal fascicular arrangement of the cells was disorganized; a few cells of intensely eosinophilic and rarely “‘bubbly” cytoplasm could be seen, morphology suggestive of cellular degeneration; furthermore, there were areas containing only a few cells, the others seemingly having been lost and replaced by an amorphous eosinophilic substance (Fig. 1). No pronoun~d differences were seen in the zona reticularis between control and experimental animals. However, in the adrenals of the experimental group, the characteristic reticular organization was lost and instead cells were arranged homogeneously. In addition, the vesicular spaces could not be clearly seen. In the adrenal medulla, the only aberrant histological characteristic seen in the experimental and not the control adrenals was the presence of a few cells of lightly staining, almost clear cytoplasm. All the above-mentioned characteristics are suggestive of adrenal degeneration in the experimental group of animals. The abnormal histology was more pronounced in the adrenals of the experimental females, indicating that the females were more vulnerable to the prenatal exposure to high levels of circulating maternal glucocorticoids. Plasma corticosteru~e
HPA axis function was significantly altered in offspring of mothers that had received ACIH injections during pregnancy (Fig. 2). Two-way ANOVA showed statistically significant treatment and sex effects on both basal and stress-induced plasma levels of corticosterone: F(1,43)=15.43, P
hS4
M . Famcli ef ol.
respective effects of sex. On the other hand, no statistically significant treatment-sex interac Zion was found for either basal or stress-induced corticosterone levels. Subsequent paired i-te sting revealed that basal plasma corticosterone levels were higher in both male and female experimc entat animals, compared to the controls. In contrast, both male and female experimental animals attz lined lower corticosterone levels than the controls, after exposure to a stressful stimulus. Brain type II glucocorticoid receptors MOstatistically s~~~f~~nt differences were found either in the concentration fBmax>or the aff linity (Kd) of the gkcocorticoid receptor in the brain of the offspring as a result of the experimr Sntat treatment (Table I).
HPA development in offspring of hyperactive HPA mothers
655
Fig. 1. Effect of ACTH administration to the pregnant dam on the histology of the adrenals of the offspring when adult. Adrenals of control (la, b) and experimental (IIa, b) female animals. The bars delineate the limits of the fascicular zone (Ia, IIa): note the reduction in its thickness in Ila. Also note the disorganization of the normal fascicular arrangement of the ceils in the adrenals of the experimental animals (IIb). Arrow points to an area where cells seem to be missing (Iib). H & E. Ia and IIa X100; Ib and IIb X400.
Brain monoamines
Treatment of the pregnant dam with ACTH apparently affected the brain of the developing embryos in a permanent fashion, since differences in monoamines were found in the brains of the offspring when they reached adulthood (Tables 2 and 3). Two-way ANOVA showed a statistically si~ificant treatment effect on striatal DA [F(1,16)= 17.93, P=O.OOl] and DOPAC [F(l,16)=20.97, P=O.OOl] levels and a significant sex effect [F(1,16)=8,71, P=O.Oll], on cortical 5-HIAA levels. A significant treatment-sex interaction was found for striatal DOPAC [F(1,16)=5.49, P=O.O39] and 5-HIAA[F(1,16)=5.68,P=O.O36],as wellascortical5-HIAA [F(1,16)=8.44,P=O.O12]. Subsequent t-testing revealed that DA was decreased in the striatum of both experimental males and females, while DOPAC was decreased in the striatum of only the experimental males. On the other hand,
M. Fameli et al.
: e
1
I
HPA development
657
in offsp~ng of hyperactive HPA mothers
w
Control
c]
Experimental
I
BISll
After
Bad
After
ItWCP
strCss**
level**
strcss-
Fig. 2. Plasma levels of circulating corticosterone under basal conditions and after exposure to stress, in male and female control and experimental animals. Values represent means?S.D. Differences between experimental and control animals: *P
Bmax(fmoles per mg protein) Male Group Control Experimental
Female
Male
Female
Mean
S.D.
n
Mean
S.D.
n
Mean
S.D.
n
Mean
S.D.
n
474 395
117 104
5 5
409 380
117 98
5 5
3.35 3.49
1.53 1.72
5 5
3.62 3.47
1.82 1.75
5 5
5-HIAA was increased in both the striatum and the cerebral cortex of the experimental Other statistical significant differences were not found in brain monoamines.
maies.
DISCUSSION The hyperactivity of the maternal HPA axis during pregnancy (induced by injections of ACTH) profoundly affected the development of the HPA axis of the offspring. The active factor mediating the effects could be corticosterone, since it has been shown to circulate at high levels in the fetus, after exposure of the mother to stress.12,15 The consequences of the prenatal treatment were manifested at all levels of the HPA axis. In the brain, changes were found in the levels of dopamine, 3,4-dihydrox~henyla~tic acid and 5-hydroxyindoleacetic acid, suggesting that the prenatal treatment acted centrally to affect HPA function. Changes in serotonergic16-1s and catecholaminergi&27 activity as a result of prenatal stress have also been reported by others. Since the CNS of the experimental offspring was affected, one would expect altered levels of type II glucocorticoid receptors in the brain, since it is known that they play a key role in controlling the stress response. 21 Indeed it has been shown that there are fewer dexamethasone binding sites in the hippocampus of prenatally stressed female rats. 29 However, adrenalectomy abolished the difference29 and in our experiments we used cytosol from adrenalectomized animals for the determination of type II glucocorticoid receptors. This could explain the fact that we found no difference in dexamethasone binding sites in the brain between the experimental and control animals. The effects of the prenatal treatment on the brain could have acted in such a way as to program the basal level of adrenal function at a setting higher than the normal, resulting in high resting levels of circulating corticosterone, in spite of the histological appearance of the adrenal zona fasciculata, which showed signs of degeneration. It appears that the portion of adrenal tissue which remained intact, functioned more actively. Weinstock et al. 29 have also found higher resting levels of serum corticosterone in adult female rats exposed to a prenatal stress. The continuous hyperactivity of the
adrenal seems, however, to have led to its exhaustion. Adrenal weights were reduced and adrenat histology was suggestive of degeneration and the results were more pronounced in the females. A reduction of adrenal weight as a result of prenatal stress has also been reported by Suchecki and Paiermo Net~.~s The exhaustion of the adrenal cortex could be responsible for the inability to respond ade4uat~Iy to the ~halleu~~ of a stress. The experimentai animals thus had a defective emergency response, attaining lower corticosterone levels than the controls. Pollardi also found that offspring of stressed mothers had a defective emergency response. It is known that in humans, stress can precipitate depressive illness in predisposed individuals” and that elevated plasma giucocorticoids are commonly found in this iilness.‘4 Since antidepressants act by modif~ng serotonergi~ and dopami~~ergi~ activities, a defect in these neurotransmitter systems is considered to be one of the underIying bioIogicaI etiologicaf factors in depr~ss~o~.~~ Furthern~ore, females, both humans~ and rats.” are more vulnerable to the development of depression. Based on the above (with all due caution as to the relevance of animal models to the respective human situations), the experimental animals of our study can be considered to represent. an animal model of individuals predisposed to deveiop depression. Not only do they have high levels of ~ir~uIating glucocorfi~oids~ but they also tack an effective emergency response and are thus unable to adapt, since adaptation is mediated by the stress-induced rises of corticosferone3 Indeed it has been reported4 that. in rats, prenatal stress results in increased vulnerability and decreased habituation to stressful stimuli. On the other hand, the inability to successfully cope with or adapt to stress is known to be able to lead not only to altered affective states such as depression, but also to the deveIopment of ulcers, heart disease and hormonal imbalances.
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