0278 - 6840/93 aJ24.00 8 1992Fwgamn~sUd

FUNCTIOlVAL EFFECTS OF GLUCOCORTICOID EXPOSURE DURING FJSTAL LIFE

TOMOKO FUJII, MATSUE HORINAKA AND MAMORU HATA

Department of Pharmacology, Teikyo University School of Medicine, Itabashi, Tokyo, Japan.

(Final form, April 1992)

Abstract

Fujii Tomoko, Matsue Horinaka and Mamoru Hata: Functional Effects of Glucocorticoid Exposure During Fetal Life. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1993, 17:(2) 279-293. -

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4.

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Pregnan:, rats were exposed .;o hydrocortisone in a dose of 10 mg/kg on days 9-11 or days 13-15 of gestation. The offsprings born to these mothers were observed for their behavioral development and the response to kainic acid during the infantile ;Jeriod. The response to kainic acid was assessed by the frequency of we'_-dog shakes behavior and limbic seizures. The growth rate in the infantile offspring of the 13-15dHc-F, group showed a slight but significant decrease. All the 13-15dHc-F, rats exhibited the rearing activity in an open- field at 17 days of age, earlier than in the contols. The ambulatory activity in the 9-lldHc-F, rats showed a significant decrease at 15 and 17 days of age, whereas no change was shown in the I)-lldHc-F, rats. The frequency of the wet-dog shakes during the 60 minutes observa- tion after the S.C. injection of 9 mg/kg kainic acid was signifi- cantly low in both the 9-lldHc-F I and 13-15dHc-F1 groups as compared with that in the controls when tested at 25 days of age. The decrease in the response to kainic acid was slightly greater in the 9-lldHC-F; rats. The frequency of seizures with forelimb clonus and rearing during the 60 minutes observation in the 13-15dHc-F, was less than that in the controls I whereas no significant difference in the frequency of seizures between the 9-lldHc-F1 and paired control groups was noted. The second generation rats raised from the 9-lldHc-F>rats by brother -sister mating showed a decrease in the frequency in the kainic acid-induced wet-dog shakes as shown in the F, offspring. No change in the response to kainic acid was shown in the 13-15dHc-F2 rats.

Keywords: F1 and Fn generations, fetal exposure, hydrocortisone, kainic acid, open-field behavior,

Abbreviations: exposure to He exposure to Hc on days 13-15 of (Gel, hydrocortisone (Hc).

wet-dog shakes.

on days 9-11 of gestation fQ-LldHcf, gestation (13-15dHcf, glucocorticoid

279

280 T. Fu]U etaL

Introduction

Evidence showing the functional effects of maternal or even paternal

exposure to drugs on the offspring has been accumulated. The authors

have already reported that various central nervous system acting drugs,

such as haloperidol, chlorpromazine, imipramine, phenobarbital,

ethosuximide, valproate or phenytoin given to pregnant rats induce

functional alterations in the adult offsprings. Moreover, changes in the

vaginal opening (Fujii et al., 1987), basal body temperature (Fujii et

al., 19871, open-field behavior (Fujii et al., 1987: Nakanishi and

Fujii, 1990), or the response to haloperidol (Fujii et al., 1982) or to

chlorpromazine (Fujii and Ohtaki, 1986; Fujii, 1988) were exhibited in

the second generation offspring raised from the rats those exposed

prenatally to these drugs. Therapeutic uses of Gcs have been well known

to be aimed at relieving various symptoms with no limitation of the age

of patients. Recent progress in understanding of the direct action of

Gcs on the brain has been facilitated by the studies on the distribution

and function of Gc receptors and Gc receptor expression in the brain

(Funder and Sheppard, 1987; Evans and Arrizal 1989; McEwen and Gould,

1990). Evidence of the rapid induction of Gc receptor mRNAs in the

brain by bodily conditions in the animals (Nichols et al., 1988) is also

important. In addition, the highest distribution of Gc receptors was

observed by several investigators in the hippocampus where the

regulatory role for the behavioral performance is suggested. However,

there are few reports on the functional effect of maternal exposure to

Gcs on the offspring.

The present experiment was conducted to study the functional effects

of maternal exposure .to hydrocortisone on -the offspring in the rat. A

particular concern was paid upon the relation of the gestational stage

of exposure to the hormone. Neurogenesis in the cerebral cortex and

hippocampal regions has been reported to begin on days 11-12, reaching

the plateau on days 13-14 in mice (Rodier, 1980). The fetal life in

mice is average 19-20 days instead of 21-22 days as for rats.

Therefore, in the present study Gc exposure was performed on the two

different stages during gestation, i.e., days 9-11 of gestation as the

stage of neural element migration and days 13-15 of gestation as the

stage of neurogenesis.

Methods

Animals

Pregnant Wistar-Imamichi rats were purchased from Animal Research

Foundation (Saitama) and the animals were injected subcutaneously (s-c.1

Glucocorticoideqosure duringfetalhfe 281

with 10 mg/kg hydrocortisone sodium succinate on days 13-15 or days 9-11

of gestation. Control rats received vehicle alone. The day on which

sperm was present in the vaginal smear was desiqnated as day 0 of

pregnancy. Litter size was adjusted to 14 pups at 3 days of age. All

rats were kept in a temperature controlled room (22+3'JC) with a lighting - schedule of 14 hr light (06:00-2O:OO) and 10 hr darkness. They were

supplied with a stock diet and water g libitum. The pups were weaned

at 21 days of age. In the presnt experiment, we did not use cortico-

sterone which is the species-typical Gc of rats because of its solubi-

lity. The effect of exposure of fetuses to propylene glycol or ethanol

as vehicle of Gc was avoided. Furthermore, as a preliminary experiment

to assess the transmission of some altered functions developed by Hc

exposure in the first generation offspring, the second generation was

raised from the 9-lldHc-F,, 13-15dHc-FI and vehicle-F1 rats by brother-

sister mating and their response to kainic acid was tested.

Drugs

Hydrocortisone sodium succinate (Solu-Cortefe , Upjohn Japan) was

purchased from a standing supplier and dissolved with distilled water.

Kainic acid (Sigma) was purchased from Iwai Chemical Co., Ltd. Kainic

acid solution was prepared before its use by dissolving in distilled

water, and the pH was ajusted to 7.0 with l/lON NaOH solution.

Open-Field Behavior

Open-field behavior was observed during the infantile period every

other day beginning at 15 days of age till 21 days of age. The open-

field apparatus with a diameter of 27.5 cm at the bottom and 30 cm at

the top with a hight of 11 cm was divided into 7 regions with a center

circle. Parameters observed were ambulation (number of square crossed),

rearing (number of times each rat raised its forepaws off the floor of

the apparatus), grooming, head shakes, urination and defecation. The

latency to the first line crossing was recorded at each test. In order

to avoid hypothermia of pups due to the isolation from mothers, open-

field behavior was limited to 2-minutes observation.

The Resoonse to Kainic Acid

Hc-F, rats and paired controls were injected S.C. with 9 mg/kg kainic

acid at 25 days of age and the time course changes in the frequency of

wet-dog shakes and limbic seizures were counted 10 minutes apart for 60

minutes. Only one test was applied to each rat.

Data Analysis -

Two-way analysis of variance (ANOVA) was used to assess the effect of

282 T. Fujiietd.

Hc treatment. Average values of data are

test. A probability level of 0.05 or greater

Results

Growth Rate

compared by the Student t-

was considered significant.

As shown in the body weight curves in Fig.1, growth retardation was

manifested from 15 days of age afterward in the 13-15dHc-F, rats (ANOVA,

P< o:osj. Growth rate in the 9-lldHc-F , rats was slightly smaller at 15

days of age but it was accelerated at 17-21 days of age, though it was

not statistically significant.

DY nc *.,e 0 .

FW.1. 0 .

SC T.

7 13 15 17 19 21

Age in Days

Fig 1. Body weight curves in the 13-15dHc-F, male and female rats and controls.

Open-Field Behavior

The 9-lldHc-F1 male and female rats showed a delay of the development

of ambulatory behavior in the open-field at 15 and 17 days of age

(Fig.2). However, the ambulatory activity returned to the control level

at 21 days of age. No change in the ambulatory activity was observed in

the 13-lSdHc-F, rats (Fig.2). Ninty-five -to 100% of the Wistar-Imamichi

strain rats used in the present experiment shows rearing activities in

the open-field at 19 days of age as presented in Fig 3. All the 13-

15dHc-F, male and female rats exhibited rearing activities in the 2

minutes observation already at 17 days of age (Fig.3), while no

significant change in the rate of rearing was demonstrated between the

9-lldHc-F, and control rats at 17 days of age.

Glucocorticoidexposureduring fetalbfe 283

g-116 MC-F1 11-156 K-F,

ts-

20 .

d E ‘*

‘$ lo-

N 5’

2 OL

2 25’

1 20-

Q 8,s. 10.

s-

o-

“.* P I5 I7 2,

15 I7 21

Age in Days

Fig 2. Ambulatory activity in the 9-lldHc-F1 and 13-15dHc-Fl rats in the open-field. Each value represents the mean+S.E.M. of 12-14 rats.

*I PiO.05 vs. age-matched controls.

9-lid HZ-F1

0 Vehicle = RC

Age in Days

Fig 3. Incidence of rearing in the same rats as in Fig 1 of the 9- IldHc-Fl and 13-lSdHc-F1 aroubs in the open-field. **, PC 0.01 vs. age-matched controls (vehicle):

Response To Kainic Acid

The frequency of the wet-dog shakes induced by kainic acid decreased

in both the 9-lldHc-F, and 13-15dHc-F, rats. The decrease in the

response to kainic acid in the 9-lldHc-F, rats, both male and female,

was greater than in the 13-15dHc-F, rats (Figs. 4, 5).

284 T.FuJii etaI.

P $ ,,:

0 0” l HC 40 l %&df!c ,;’

,/

10 20 30 ,o 50 60

Fig 4. Time course of the frequency of wet-dog shakes following S.C. administration of kainic acid in the 9-lldHc-FI and 13-15dHc-F, male rats. The rats were tested at 25 days of age. Each point represents the mean+S.E.M. of 12-14 rats. *,p< 0.05 vs. DW-FI controls. ANOVA for - the 9-lldHc-F1, PC 0.05.

9-lid "C-F, 'or 13-15d "C-F, I

Time after K4lnic Acid (Mln)

Fig 5. Time course of the frequency of wet-dog shakes following S.C. administration of kainic acid in the littermate female rats in Fig 4. Each point represents the mean+S.E.M. of 9-12 rats. ANOVA for the 9- IldHc-FL , P< 0.05. *,P< 0.05-v~. DW-FI rats.

The response to kainic acid was tested in the second generation rats of

the 9-lldHc-F, and 13-15dHc-F, parents. _!:e 9-lldHc-F, rats in both

male and female showed a decrease in the frequency of the wet-dog shakes

after adminsitration of 9 mg/kg kainic acid (Figs. 6, 71, though the

significant decrease was observed only at 50 and 60 minutes after the

treatment with kainic acid.

Glucocorticoid exposun during fetal life 285

9?Idl-lC-Fz 13-l?% HC-F,

Tii afw Kaid &id (Mm)

Fig 6. Time course of the frequency of wet-dog shakes following S.C. administration of kainic acid in the 9-lldHc-FI and 13-15dHc-Fa male rats. The rats were tested at 25 days of age. Each point represents the mean+S.E.M. of the number of rats in parenthesis. *,p< 0.05; **,p< 0.01 vs. BW-FI.

Q-l ld HC-Fz Female

13-156 HC-h

iii alter Kainiic A& (Min)

Fig 7, Time course of the frequency of wet-dog shakes following S.C. administration of kainic acid in the littermate females of the rats in Fig 5. Each point represents the mean+S.E.M. of the number of rats in parenthesis. *,P< 0.05; ***rP< 0,001 vE. DW-Fr.

Discussion

The present experiment was focused on the functional effects of Gc

exposure on the fetal hippocampal region and cerebral cortex.

Therefore, the endpoint for the assessment used was the locomotor

activity in an open-field and the response to kainic acid which posses-

ses a high density of its receptors on the cells of the hippocampus.

286 -r.Fuytefc4L

The results obtained in this experiment demonstrated that maternal

exposure to hydrocortisone in the rat for 3 days at either stage of the

migration of neuronal elements or of neurogenesis mainly in the cerebral

cortex, amygdala and hippocampal pyramidal cells affected the

behavioral development and the response to kainic acid in the offspring.

Glucocorticoid Receptors in the Brain

Glucocorticoid (Gc) receptors are widely distributed in the brain and

the hippocampus is a principal neural target tissue for Gcs with high

concentraions of the receptors. However, physiological role of the Gc

receptors in the brain is still obscure. Reul et al. (1989) studied on

the gene expression of Type I Imineralocorticoid, high affinity gluco-

corticoid) and Type II (classical glucocorticoid) receptor mRNA in

various tissues and clarified that Type I mRNA is high in the hippo-

campus, colon, and heart, and Type II mRNA was high in the liver,

thymus, and brain. Adrenalectomy induces a transient increase in both

hippocampal Type I and Type II mRNA.

There are few reports on the ontogenetic appearance of Gc receptors in

the hippocampus or other regions of fetal brain. Kalinyak et al. (1989)

found that Gc receptor mRNA is detected in rat brain at 20 days in fetal

life. On the other hand, adrenal cells have been indicated to appear at

days 13.5-15 of fetal life in the rat (Roes, 1967; Sugihara, 1977).

With these findings, it can be assumed that fetal Gc may play some role

not only in adaptation to stress during parturition but also in the

brain development. Cells in the process of migration and/or proli-

feration in fetal body, in a highly active state, could be affected by

exogenous Gc to a greater extent as compared with those in resting or

mature condition.

Numerous neurochemical and electrophysiological studies demonstrate

that the hippocampus is extremely sensitive to the presence of Gcs (Reul

and De Kloet, 1985). Sapolsky and his colleague have suggested that Gcs

appear to induce a general metabolic vulnerability in hippocampal

neurons, impairing their capacity to survive varied insults (Sapolsky,

1986; Sapolsky and Pulsinelli, 1985). Armanini et al. (1990) suggested

that Gcs endanger hippocampal neurons by enhancing glutamatergic signals

and/or enhancing vulnerability to such signals. Therefore prolonged Gc

exposure can ultimately be deleterious. Indeed, when exposure to Gcs is

prolonged in therapeutic uses consequently steroid diabetes, fatigue,

myopathy, hypertension, and immunosuppression develop. The findings

that chemical adrenalectomy reduces hippocampal damage induced by kainic

acid (Stein and Sapolsky, 1988) may support the ability of Gcs to

compromise neuronal viability in the hippocampus.

Rat hippocampal cells prepared from embryos at days 17 and 21 of

Glucocorticoidexposureduringfetallife 287

gestation are capable of responding with depolarization to nanomolar

concentrations of muscimol, a GABA agonist, suggesting that a receptor

mediated response exists (Fiszman et al., 1990). The time of neuron

origin in all areas of rat hippocampal region was examined by Bayer

(1980) with "H-thymidine autoradiography. The progress of neurogenesis

in the hippocampal region begins from embryonic days 15-16 through days

17-19. In his experiment, the presence of sperms in the vaginal smear

was designated as embryonic day 1 instead of day 0 in our experiment.

In the present experiment, exposure to hydrocortisone on days 9-11 of

gestation before the peak of neurogenesis in the hippocampus and

cerebral cortex (Rodier, 1980) resulted in more marked changes in

behavioral development and the response to kainic acid.

Response to Kainic Acid

Kainic acid is a rigid glutamate analogue and it binds with cells in

the hippocampus which possesses glutamate receptors. Behavioral mani-

festations after kainic acid treatment are characterized by scratching,

wet-dog shakes, rearing, forelimb clonus and generalized clonic and

tonic limbic seizures. Appearance of the wet-dog shakes behavior

following administration of kainic acid has been considered as the

result of the binding of kainic acid to high affinity components in the

limbic system (Berger et al., 1984). Kainic acid binding sites are

associated with the mossy fiber terminals which project upon the

pyramidal neurons of the CA3 region in the hippocampus (Represa et al.,

1987). Albala et al. (1984) observed that rat pups at 15-18 days of age

do show early generation of convulsions with few wet-dog shakes after

kainic acid administration, and that selective lesions consisting of

neuronal cell loss in the hippocampus, amygdala and pyriform cortex were

only shown in puberscent and adult rats. The necrotic lesion was well

associated with the limbic seizures. Hata and Fujii (1991) also have

examined the age difference in the response to kainic acid in Wistar-

Imamichi rats and found that the wet-dog shakes behavior and limbic

seizures can be developed following the systemic administration of

kainic acid after 3 weeks of age. Therefore, in the present experiment,

the response to kainic acid was assessed at 25 days of age.

The wet-dog shakes behavior can also be induced by administration of

various drugs. Systemic administration of a precursor of serotonin, 5-

hydroxytryptophan (5-HTP) is well known to induce wet-dog shakes

behavior (Bedard and Pycock, 1977) and it induces an increase in

cerebral serotonin concentration. Yoshida et al. (1986) observed that

intraventricular administration of [D-Ala2,Met']enkephalinamide is capable

of inducing wet-dog shakes and epileptic discharges in the hippocampus.

Wet-dog shakes elicited by kainic acid, however, differ from that

288 T. Fujii et al

produced by 5-HTP in the features with the onset of frequent scratching

and with limbic seizures. The frequency of 5-HTP induced wet-dog shakes

was lesser than that induced by kainate (Taguchi and Fujii, 1987).

Behavioral Development

In the present experiment, the offspring exposed prenatally to Hc on

days 13-15 of gestation showed accelerated development of rearing

behavior without reduction in the scores of ambulation at 17 days of

age. This was earlier than the time of its appearance in normal rats.

Rearing behavior has been considered to reflect increases in

dopaminergic activity primarily in the nucleur accumbens (Costa11 et

al., 1977). In the 13-15dHc-F, rats, ambulatory score did not show any

change in spite of the increased number of rearing, suggesting that

locomotor activity in these rats increased. Behavioral performance has

been considered by many researchers as to be well related to the

functional activity of the frontal cortex, hippocampus and amygdala.

Moreover, spontaneously generated behavior can be very sensitive to drug

treatment and can even indicate the severity of the effects as reported

previously on the several CNS acting drugs (Fujii et al., 1987, Fujii

and Kusama, 1991; Nakanishi and Fujii, 1990).

Catecholaminergic systems, particularly dopaminergic systems play an

important role in the mediation of general activities in young animals

in controlling the spontaneous activity in rodents (Barrett et al,,

1982; Beninger, 1983; Camp and Rudy, 1987; Pichler and Pifl, 1989).

Cholinergic neurons in the hippocampus and cortex also correlated with

the level of locomotor activity (Day et al., 1991). It is of interest

that the exposure to Hc in the rat on days 9-11 of gestation before the

stage of the neurogenesis in the cerebral cortex and hippocampus

suppressed the development of spontaneous behavior in an open-field to a

greater extent as compared with that seen in the rats exposed to Hc on

days 13-15 of gestation. The rats exposed to Hc on days 13-15 of fetal

life showed a slight but significant delay of the body growth. There

are several reports on the growth retardation after Gc treatment in man

and animals. This inhibition is probably due to its catabolic action.

However, it is difficult to propose the long-term metabolic effect of

the short-term exposure to Gc during fetal life. Gc appears to reduce

the growth hormone secretion (Wehrenberg et al., 1990).

Maternal Exposure to Glucocorticoid

There are several reports on the effect of maternal exposure to Gc

during pregnancy on the fetal brain. Velazquez and Roman0 (1987)

treated pregnant mother rat at 17, 18 and 19 days of gestation. They

observed the pups at birth or at 6 or 12 days of postnatal age and found

the decreased DNA content and accelerated decrement of the external

granular layer in the cerebellum. Recent observations by Slotkin and

his colleagues (1991) have demonstrated that dexamethasone given to

pregnant rats on gestational days 17, 18 and 19 resulted in an enhance-

ment of the binding of ($B]desmethylimipramine (DMI) in the midbrain,

brainstem and in cerebellum when 0.05 mgJkg was given but without change

in the growth. At a higher dose, 0.2 mq/kg that elicited moderated

growth impairment induced an initial enhancement and subsequent deficits

in binding of /IQ]DMI in the midbrain and brain stem. Cc-induced

behavioral changes have been observed in rats by Wolkowitz and his

coleagues (1986). Seven daily injections of 10 mg/'kg of corticosterone

induced a significant increase in the vertical and ambulatory locomotion

with an increase in the caudate homovanillic acid, suggesting the

increased central dopaminergic activity. Lee and his coleagues (1989)

reported that administration of dexamethasone 1 hr before kainic acid

administration in adult rats potentiated the wet-dog shakes and severity

of seizure activity. In the present results, however, the response to

kainic acid decreased significantly in all the rats born to &z-exposed

mother rats when the frequencies of wet-dog shakes were assessed. The

suppression was slightly larger in the 9-lldHc-FI rats and this altered

feature was transmitted to their second generation. It might be

possible that acute effect of Gc priming in modulating the effect of

kainic acid differs from the long-lasting effect of prenatal exposure to

Cc. The present results suggest a possibility that the development of

receptors for kainic acid might be altered in the Hc-FI and Fr rats,

Transgenerational Effect

This is the first report showing the transgeneration of the altered

response to kainic acid developed in the FI rats after a short-term

exposure to hydrocortisone during gestation. The decreased incidence in

kainic acid-induced wet-dog shakes behavior shown in the 9-lldHc-Flrats,

male and female, was transmitted to the second generation. Although its

mechanism is unknown at present, evidence showing the transgeneration of

altered function developed in the F1 animals after maternal exposure to

drugs or chemicals has been accumulated (Reimers and Sluss, 1978; Beach

et a1.,1982; Fujii et a1.,1982, 1987; Fujii,1988). Even maternal

endocrine disorders such as calcium regulatory system (Fujii, 1978;

Fujii et al., 1980, 1988) or glucose metabolism (Baranov et al., 1988)

can induce alterations of the regulatory function of blood calcium or

glucose in the F, rats and the altered features have been found to be

heritable to subsequent generations. Though the mechanism of these

findings have not been defined at present, there are some hypothetical

interpretaitons made on the trangenerational effects (Campbell, 1991;

Gruenert and Cozens, 1991).

290 T.Fujfl etaL

Conclusion

Maternal exposure to hydrocortisone only for 3 days during the

migration of neural elements (days 9-11 of gestation) or during the

neurogenesis (days 13-15 of gestation) in the fetal brain clearly showed

to affect the growth, behavioral development, and functional development

of hippocampal regions in the rat when assessed using 3 measures, body

weight, open-field behavior and frequencies of kainic acid-induced wet-

dog shakes and seizures. The decreased wet-dog shakes response to

kainic acid in the 9-lldHc-F, rats was transmitted to the Fz generation,

while no change in the response to kainic acid was observed in the 13-

15dHc-Fn rats.

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Inquiries and reprint requests should be addressed to:

Dr. Tomoko Fujii Department of Pharmacology Teikyo University School of Medicine 2-11-1, Kaga, Itabashi-ku, Tokyo 173 Japan.