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Am J Physiol Regul Integr Comp Physiol 279: R1899-R1909, 2000;
0363-6119/00 $5.00
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Vol. 279, Issue 5, R1899-R1909, November 2000

Effects of prenatal dexamethasone on spatial learning and response to stress is influenced by maternal factors

Tiffini Brabham1, Andrew Phelka1, Carrie Zimmer1, Aleda Nash1, Juan F. López1, and Delia M. Vázquez1,2

2 Department of Pediatrics, Endocrine Division and 1 Mental Health Research Institute, Department of Psychiatry, University of Michigan, Ann Arbor, Michigan 48109


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study investigated the effect of prenatal dexamethasone (Dex) exposure on early perinatal events, hippocampal function, and response to stress. Pregnant rats received Dex in the evening water (2.5 µg/ml) or tap water (Veh) from gestational day 15 until delivery. On the day of parturition, pups were randomized, cross-fostered, and reduced to eight or nine per dam. Four groups resulted: Veh-Veh (offspring exposed to Veh in utero, rearing mother treated with Veh during gestation), Veh-Dex, Dex-Veh, and Dex-Dex. Spatial visual memory was evaluated with the Morris water maze. The corticosterone response to restraint stress was examined, and the expression of hippocampal glucocorticoid and mineralocorticoid receptors mRNA was determined by in situ hybridization. Exposure to Dex caused restlessness in mothers, low birth weights, and poor weight gain in the offspring. The Dex-Dex males had impaired spatial learning, inability to rapidly terminate the adrenocortical response to stress, and decreased hippocampal glucocorticoid receptor (GR) mRNA expression. In contrast, Dex-exposed animals reared by Veh-treated mothers had adequate spatial learning, enhanced glucocorticoid feedback, and increased hippocampal GR mRNA. We conclude that the environment provided by a healthy mother during the postnatal period can prevent the detrimental effects of prenatal Dex administration on cognition, GR mRNA expression of the hippocampus, and the quality of the stress response.

rats; animals; development; hippocampus; glucocorticoid receptors; mineralocorticoid receptors; mRNA


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

STUDIES IN VARIOUS SPECIES, including non-human primates, demonstrate that early exposure to synthetic glucocorticoids retard both fetal and placental development (8, 22). Although clear risk for brain maldevelopment, hypertension, and behavioral and cognitive consequences have been reported in animal studies (2, 4, 6, 9), less is known about long-term effects on other functions directed by the brain. Despite these concerns, there are certain maternal and/or fetal medical conditions that are treated with synthetic glucocorticoids during early gestation (18). Long-term effects of in utero synthetic glucocorticoid exposure may be mediated by direct effects of dexamethasone (Dex) on general cell processes related to central nervous system development and/or effects on specific fetal brain structures. An indirect effect on the offspring is also possible, because in the process of exposing the fetus to synthetic glucocorticoids, the mother is also subjected to the effects of glucocorticoids, which may alter maternal physiology during and after gestation. This becomes an important issue, because glucocorticoids may alter maternal health and behavior in ways that may compromise the postnatal offspring development.

The direct effects of glucocorticoids on brain development are mediated through corticosteroid receptors: the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). Dex, however, binds exclusively to GR (36), whereas MR has a particularly high affinity for corticosterone and also recognizes the mineralocorticoid aldosterone. The brain distribution of these receptors is distinct. The GR is identified throughout the brain (neurons and glia), whereas both GR and MR are highly expressed in limbic structures, such as the hippocampus. Interestingly, these receptors follow a unique pattern of expression within the hippocampus during fetal and postnatal life, a pattern that can be disrupted by adverse events or pharmacological treatment (3, 38, 44-46). Of interest is the fact that most recently it was recognized that maternal behavior is capable of altering GR development (19, 26). Specifically, increased maternal care increases the expression of GRs in the hippocampus and alters the hippocampal function in the animal. Therefore, maternal care could theoretically modify the long-term effects of prenatal insults. This possibility has not been explored in previous studies addressing adverse effects of prenatal Dex treatment.

In a series of studies presented here, we investigated an area that has not been studied, namely, the long-term effect of prenatal Dex treatment on hippocampal function by testing the adrenocortical response to restraint stress, and cognitive capacity using the Morris water maze. In an attempt to determine whether the effects of gestational treatment were mediated prenatally or were in part due to different rearing by a mother also exposed to the brain effects of glucocorticoids, we used a cross-fostering design. We compared the adult offspring of mothers treated with Dex during gestation but reared by nontreated mothers and those animals born to nontreated mothers reared by Dex-treated mothers. The results of the tests were correlated with the GR and MR mRNA expression in the hippocampus, because within the hippocampus, the balance of GR and MR is critical for two functions: the inhibition of the stress-induced activation of the limbic-hypothalamic-pituitary-adrenal axis (LHPA) and visual spatial cognition (8). We hypothesized that in utero Dex treatment would alter these two functions directed by the hippocampus. We further hypothesized that offspring reared by mothers that were treated with Dex during gestation and until parturition would worsen these outcome measures in animals that received Dex in utero. Our data indicate that the long-term impact of in utero Dex treatment is indeed dependent on several factors, including in utero drug exposure, factors intrinsic to the mother-pup-rearing environment, and sex of the offspring. Moreover, our study suggests that a rearing environment provided by a healthy mother can alter the biological trajectory dictated during prenatal life.


    MATERIAL AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Sprague-Dawley female rats (Charles Rivers, Wilmington, MA) were mated in our animal unit and maintained in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The animals were kept under constant temperature (25 ± 2°C) and lighting (14:10-h light-dark cycle) condition. Rats were provided with rat chow (Wayne Rodent Blox, Allied Mills, Chicago, IL) and water ad libitum. Sperm detection on vaginal smears marked the first day of pregnancy. On day 15 of gestation, pregnant females were housed individually in clear polycarbonate cages (Nalgene, 24 × 45 × 20 cm) to start the drug treatment.

Drug treatment. Dex (Sigma, St. Louis, MO) was dissolved with ethanol and added to the drinking water starting on gestational day 15 (GD 15) until the day of delivery. Guided by a previous study (13), the dose of 10 µg/ml Dex was administered daily from GD 15 until the day of delivery. However, this treatment resulted in a high incidence of fetal abortion, thus the dose was lowered to 5 µg/ml. This dose resulted in 50% maternal mortality on spontaneous vaginal delivery. On the basis of this outcome, the dose was once again reduced, to 2.5 µg/ml. This dose suppressed ACTH and corticosterone levels in five pregnant rats that were tested at 1700 (data not shown, and offspring were not used for the actual experiment). Thus the data reported here are from the offspring of a total of 24 pregnant rats that received 2.5 µg/ml Dex-0.03% ethanol added to the evening water (2 h before lights off and until 2 h after lights on: 1700-0700). Control animals received tap water with an equivalent concentration of ethanol [vehicle (Veh) (n = 20)]. The water supplied during the day was tap water offered ad libitum for both groups.

Parturition and cross-fostering. On the day of parturition, the Dex or Veh solutions were discontinued and replaced with tap water. The litters were randomized, cross-fostered, and reduced to eight or nine pups per dam by day 3 of life. Four groups of offspring resulted: 1) Veh-Veh (offspring exposed to Veh in utero, rearing mother treated with Veh during gestation; n = 36), 2) Veh-Dex (offspring exposed to Veh in utero, rearing mother treated with Dex during gestation; n = 42), 3) Dex-Veh (offspring exposed to Dex in utero, rearing mother treated with Veh during gestation; n = 44), and 4) Dex-Dex (offspring exposed to Dex in utero, rearing mother treated with Dex during gestation; n = 34). Each mother reared Veh and Dex in utero-treated pups. Pups were weaned on postnatal day 21 (PND 21) and grouped three to six animals per cage according to sex and age with water and food available ad libitum until testing in adulthood.

General Measurements

Daily fluid consumption was recorded according to light or dark cycle during gestation and daily during the postnatal lactation period. Maternal body weights were recorded on GDs 15, 18, and on parturition. On parturition, perinatal mortality was recorded. Weekly litter and maternal body weights were recorded when the animals were placed in cleaned cages as part of their weekly animal care routine. In addition, offspring weight gain was assessed periodically.

Behavioral Testing

Maternal care. Maternal behavior was recorded on PNDs 2, 6, and 10. Twelve dams were selected at random (6 Veh and 6 Dex). Twenty-four-hour periods were recorded using a time-lapse recorder (Panasonic, model AG-6730). The analysis of the behavior was computerized with an observer program (kindly provided by Dr. Terry Lee, Psychology Department-University of Michigan). The program recorded the starting time and duration of events from videotape. A trained observer registered the time a mother spent engaged in the following behaviors: nursing, grooming of pups, eating and drinking, running, resting. Under the parameters of the program, an event was counted as distinct if a minimum of 3 min separated the preceding event from the new event. In addition, the event was distinct only if its duration was 5 min or more. The persons scoring the tapes were not aware of the maternal drug treatments.

Morris water maze . The test was performed when animals were young adults, at 65-75 days of age. For each experimental group and for each sex, 16 animals originating from different litters were tested. The procedure was adapted from Morris et al. (31). To avoid stress from animal odors, all male animals were tested for 6 consecutive days; the room was sanitized and then females were tested. The water maze was a circular pool (140-cm diameter, 50-cm high) that was filled with water (25°C) made opaque with coffee creamer (Carnation). A clear escape platform (11-cm diameter) was placed in the middle of a quadrant determined not to be the preferred swimming site by the group of rats being tested. The platform was stationary, 1-2 cm below the water level, equidistant from the sidewall and the middle of the pool. The testing room contained numerous constant visual cues outside the tank for orientation (e.g., black or white posters of different geometrical shapes, a large circular clock, garment poles). An individual in the room that also served as a stationary extramaze visual cue monitored the behavior of the animal.

Four different starting positions were equally spaced around the perimeter of the pool. The time (or latency) to find the submerged platform was registered. A block of four was performed per day, for 5 consecutive days. Free swim trials lasting 120 s were performed as by others (8, 27); that is, on the 5th day (trial 21), after the last trial of the day, and on the 6th day, when the four block trials were omitted (trial 22). The time spent in the quadrant where the platform was previously located was used as a measure of retention. A "cue" test was also performed on day 6 to verify that the animals did not have sensorimotor deficits.

Biological Measures

Animals naive to the behavioral testing were submitted to a 30-min acute restraint stress in the morning (0700-1000) at 65 days of age. Blood sampling was performed using the tail vein at 5, 15, 30, 60, and 120 min after the animals were restrained. A 0-min time point was also obtained before the procedure. Five to eight animals (per group, per sex), each obtained from different litters, were used for this purpose. The animals were allowed to rest for 2 wk and were then killed in the morning (0800-1000) at 80 days of age. Blood and brains were collected.

Plasma corticosterone determination. All blood was collected in tubes containing EDTA and spun at 2,000 rpm for 7 min to obtain plasma. Corticosterone was assayed with all groups represented using a radioimmunoassay as previously described (45). The antibody cross-reacts 2.2% with cortisol and <1% with other endogenous steroids. The detection limit is 1 pg/ml, and the intra- and interassay coefficient of variation is 2 and 3%, respectively.

In situ hybridization: brain tissue processing. Brains were rapidly removed, frozen in liquid isopentane (-42°C), and stored at -80°C. Subsequently, they were sectioned in coronal plane at 10 µm on a Bright-Hacker cryostat (Hacker Instruments, Fairfield, NJ) that was maintained at -20°C, and the sections were thaw mounted onto polylysine-coated microscope slides. Brain sections were stored at -80°C until processed for in situ hybridization. All groups studied were included in each of the in situ experiments.

GR and MR probes were prepared as previously described (45). The MR probe was a 347-nt fragment of our MR clone, directed against the 3'-untranslated region of the MR mRNA. The GR probe was a 544-nt fragment of a cDNA clone directed against the protein binding region and the 3'-untranslated region of the GR mRNA. We previously showed that the probes do not contain any regions of high homology, which avoids potential for cross-hybridization. Antisense riboprobes were prepared using the appropriate restriction endonucleases and transcribed using either T7 or SP6 promoter sites in presence of 35S-labeled UTP (1,000 Ci/mmol). The reaction yielded a specific activity of 1.37 × 105 and 1.42 × 105 Ci/mmol for GR and MR probes, respectively. We followed the hybridization protocol previously described (45). The slides were incubated overnight with the respective probe at 50°C. Single-stranded probe not hybridized with endogenous mRNAs was removed by incubating the sections for 30 min in 200 µg/ml solution of RNase A at 37°C. The tissue was then washed in increasingly stringent sodium chloride-sodium citrate (SSC) solutions (2, 1, and 0.5× SSC) followed by a 1-h wash in 0.5× SSC at 50°C. After this final wash, the tissue slides were organized for detection by X-ray autoradiography on Kodak XAR-5 film. The autoradiograms generated were analyzed using an automated image-analysis system (Dage camera, MAC II/IMAGE). The person analyzing the images was not aware of the treatment conditions under analysis. Hippocampal areas, corresponding to subfield 1) CA1, 2) CA2, 3) CA3-4, and 4) dentate gyrus, were digitized from a given hippocampal section. The mean optical density of the region of interest was measured at ×100 magnification. Background labeling was measured from the corresponding internal area of each section. Four sections per animal were analyzed. The mean of these was used as the individual value for an animal. Six to eight animals were analyzed per condition, per sex.

Statistical Analysis

ANOVA procedures were used with the level of significance set at P < 0.05. The maternal behavior data were analyzed by ANOVA considering treatment group and individual days of observation as independent variables. For the Morris water maze spatial learning test, the average of the sum of the four trials per day was analyzed by ANOVA considering sex, group, and days as independent variables and by repeated-measure ANOVA. Similarly, the hormonal data were analyzed independently for sex, groups, and time of response. Whenever differences were determined to be nonsignificant, the data were collapsed across the respective variable. Post hoc comparisons were made by using Fisher's protected least-significant difference (Fisher's PLSD).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

General Measurements

Consumption. To ensure that all experimental animals received an equivalent amount of Dex, the average amount of water consumed by the Veh control group guided the amount that the experimental animals received. Even though water consumption was limited at night for the Dex-treated group, the total amount of water consumed by the Dex group did not differ from the Veh-treated animals (Table 1). Both the Veh control and the Dex-treated mothers significantly increased their daytime water consumption as the pregnancy progressed (F = 8, P = 0.0009, repeated-measure ANOVA). All Dex-treated animals received an equivalent amount of Dex during 15-21 days gestation, which averaged 0.27 ± 0.02 mg · kg-1 · day-1 (means ± SD).

                              
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  		Table 1.   The amount of daily fluid intake during gestation

Maternal body weight, birth, and mortality. No significant body weight differences were observed between the Dex-treated and Veh control mothers during gestation or during the lactation period (Table 2). In both the Dex-treated and Veh-treated mothers, a significant weight decrease was observed at parturition followed by weight gain (F = 17, P = 0.0001, repeated measure). Analysis of the weight change revealed a significant treatment effect from GD 15 to GD 18 (F = 4.3, P < 0.05, see Fig. 1A). The Dex-treated mothers had a significantly reduced weight gain during this period of time compared with Veh controls. The Dex-treated mothers showed a weight gain increase during the second week postpartum (F = 3.9, P = 0.05), which coincides with an increase in eating and drinking behavior (Fig. 1B and presented below).

                              
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  		Table 2.   Body weight of mothers during gestation and lactation periods, live births, and offspring mortality during the first 3 days of life



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  		Fig. 1.   Body weight change observed in vehicle (Veh)- and dexamethasone (Dex)-treated mothers during pregnancy and postpartum period (A) and maternal eating and drinking behavior observed in Veh- and Dex-treated mothers during lactation period (B; Veh, n = 6; Dex, n = 6). The change in time spent eating and drinking from week 1 to week 2 was significantly different in the Dex-treated mothers compared with Veh controls. G, gestational day; PND, postnatal day. * P < 0.001.

There were no significant differences in the number of pups born from each of the maternal groups (see Table 2). However, Dex-treated mothers had a significant number of deaths on delivery (t = 3, P < 0.05). In addition, a significant number of pups born to Dex-treated mothers were not healthy and died within the first 2 days of life (see total deaths, Table 2).

Weight. At birth, the weights of male and female offspring born to Dex-treated mothers were significantly different from those of the Veh-treated mothers. However, male weights were not different from female weights (Dex males = 5 g ± 0.9, Veh males = 7 g ± 0.8; Dex females = 5 g ± 0.8, Veh females = 7 g ± 1; means ± SE, mother effect, F = 64, P < 0.0001; sex, F = 1, P = 0.36). Analysis of the weight progression as the animals matured revealed a sex (F = 715, P = 0.0001), group treatment (F = 57, P = 0.0001), and age effect (F = 2,170, P = 0.0001), with a sex and age interaction (F = 185, P = 0.0001). Thus poor weight progression correlated with in utero drug exposure for both sexes. Specifically, compared with the Veh-Veh groups, both male and female rats born to Dex-treated mothers had significantly lower weights when 35, 42, and 60 days old regardless of the rearing mother (see Table 3).

                              
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  		Table 3.   Weight progression of the offspring exposed to no drug or to Dex in utero. At birth, animals were cross-fostered and 4 groups resulted.

Maternal Behavior

The pattern of eating and drinking was different among treatment groups (Fig. 1B). We detected an increment of drinking and eating in the Dex-treated mothers from week 1 to week 2 postpartum (2-way ANOVA, F = 8.9, P = 0.02). The Veh-treated mothers had the greatest increment of time spent eating and drinking from days 2 to 6 (F = 16.6, P < 0.05), whereas the Dex-treated moms had a greater increment from postpartum day 6 to 10 (F = 116.9, P < 0.001). Overall, the Veh-treated mother increased the time spent in this activity steadily (Veh: day 2 = 9.7 ± 1.2%, day 6 = 20.9 ± 6.5%, day 10 = 25.4 ± 1.4%). In contrast, the Dex-treated moms spent equal time eating and drinking on days 2 and 6, but this activity nearly doubled on the last day of recording (Dex: day 2 = 13.5 ± 1.1%, day 6 = 12.2 ± 6.8%, day 10 = 30.2 ± 1%).

With the exception of eating and drinking, the ANOVAs did not reveal any other day effects for maternal behaviors. Thus, during PNDs 2, 6, and 10, Dex- and Veh-treated mothers spent a similar percentage of time nursing and grooming their young (nurse: Dex 42 ± 4, Veh 34 ± 4; groom: Dex 23 ± 4, Veh 19 ± 2, mean% ± SD; P > 0.05, see Fig. 2). However, Dex mothers maintained high levels of activity around the nest even though they were not giving direct maternal care (run: Dex 11 ± 1%, Veh 6 ± 1%; F = 5, P < 0.05). Dex mothers also rested away from the nest much less compared with Veh mothers (rest: Dex 9 ± 3%, Veh 19 ± 3%; F = 5, P < 0.005).


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  		Fig. 2.   Maternal behavior observed in Veh- and Dex-treated mothers (Veh, n = 6; Dex, n = 6). The total time spent undergoing maternal care activity (nursing, grooming) and self-care activities (eat, drink, run, rest) were recorded for 24-h periods on PNDs 2, 6, and 10. An event was counted as distinct if a minimum of 3 min separated the preceding event from the new event. *Significantly different from Veh, P < 0.05, means ± SD.

Morris Water Maze: Spatial Task

Acquisition. Three-factor ANOVA revealed statistical differences between groups with regard to the number of trial blocks needed to learn to escape swimming using visual cues (F = 149, P < 0.0001). Performance was significantly different in males compared with females within each experimental group (F = 1,074, P = 0.0001). An interaction between group and trial blocks required to learn the task was also present (trial blocks: treatment groups, F = 3.4, P < 0.0001). In addition, we also found an interaction between sex, treatment groups, and trial blocks (F = 1.8, P < 0.05).

Males exposed to Dex in utero and reared by mothers that received Dex during pregnancy learned the water maze task on day 4, whereas all other groups had mastered this task by day 3 (Fig. 3A). This was evident on the repeated-measure analysis (treatment group, F = 11, P < 0.0001; trial blocks, F = 50, P < 0.0001; and group and trial block interaction, F = 3, P = 0.0002). Those animals that were exposed to in utero Dex treatment but were reared by control mothers (Dex-Veh) were particularly quick to reach the submerged platform on day 1 and maintained a high level of performance throughout the testing period. When individual trials were analyzed for this particular group, it was evident that the latency to reach the platform for trial 1 on day 1 was significantly higher than that seen on trials 2, 3, and 4 (trial 1 = 12 ± 1.7; trial 2 = 3.2 ± 0.75; trial 3 = 4.5 ± 0.5; trial 4 = 3.75 ± 0.75; means ± SE in seconds; F = 10.6, P = 0.0001 repeated-measure ANOVA).


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  		Fig. 3.   Performance of males (A) and females (B) on the Morris water maze spatial learning task. The time to find the submerged platform on 4 swim trials was registered daily for 5 consecutive days. The average of this sum was analyzed by ANOVA considering sex, group, and days as independent variables and by repeated measure. 1) Veh-Veh (offspring exposed to Veh in utero, rearing mother treated with Veh during gestation), 2) Veh-Dex (offspring exposed to Veh in utero, rearing mother treated with Dex during gestation), 3) Dex-Veh (offspring exposed to Dex in utero, rearing mother treated with Veh during gestation), and 4) Dex-Dex (offspring exposed to Dex in utero, rearing mother treated with Dex during gestation). *Significantly different from Veh-Veh, P < 0.05, means ± SE, n = 16 per group, per sex.

All females learned the task by day 2 with the exception of the females exposed in utero to Dex and reared by mothers that received Dex during pregnancy (Dex-Dex, Fig. 3B). These Dex-Dex females mastered the task on day 3 of the trials. Similar to males, females that were exposed to in utero Dex treatment but were reared by control mothers were particularly quick to reach the submerged platform on day 2 (Dex-Veh, repeated-measure ANOVA: trial blocks, F = 133, P < 0.0001; treatment group and trial block interaction, F = 1.8, P < 0.05).

When the performance was compared between males and females, a significant difference was detected on days 1 and 2 that was dependent on the treatment group (day 1: sex, F = 9, P < 0.005; treatment group, F = 6, P < 0.005; sex and group interaction, F = 4, P < 0.05; day 2: sex, F = 7, P < 0.05; treatment group, F = 7, P < 0.0005; sex and group interaction, F = 2, P = 0.12). On day 1, the Dex-Veh males had a shorter latency to reach the platform than did the Dex-Veh females. On day 2, the Dex-Veh females had a shorter latency compared with Dex-Veh males.

Retention. The "free swim" trial performed immediately after the last trial on day 5 revealed retention differences in males only (sex, F = 7, P < 0.008, see Fig. 4, A and B). The two groups of males reared by Dex-treated mothers spent significantly less time in the quadrant where the platform had been submerged, indicating impaired memory for the location of the platform. When the retention of the task was tested on day 6, no significant differences were detected (F = 1.3, P = 0.26). Overall, the females swam less time in the target quadrant on this day compared with males (sex, F = 3.8, P < 0.05).


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  		Fig. 4.   Retention of the spatial learning task in males (A) and females (B) across the treatment groups. A "free swim trial" was performed on the 5th day (trial 21) and on the 6th day (trial 22). The time spent in the quadrant where the platform was previously located was used as a measure of retention. *Significantly different from Veh-Veh P < 0.05, means ± SE, n = 16 per group, per sex.

Stress Response: 30-min Restraint Stress

The adrenocortical response to restraint stress of the different mother-in utero treatment combinations were compared with that of control animals (Veh-Veh) in Fig. 5. ANOVA revealed statistical differences between groups (F = 7, P = 0.0001), time (F = 131, P = 0.0001), and sex (F = 32, P = 0.0001). Interactions between group and time (F = 2, P = 0.05), group and sex (F = 3, P < 0.05), and time and sex (F = 12, P = 0.0001) were also present. With few exceptions (see below), the overall pattern of activation and termination of the stress response for each of the in utero-exposed rearing groups was similar between males and females (Fig. 5, A and B).


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  		Fig. 5.   Time course of the plasma corticosterone (CS) secretion in response to a 30-min restraint stress in males (A) and females (B). The response of each individual group at a particular time point is compared with the CS response of the Veh-Veh animals. *P < 0.05, means ± SE, n = 5-8 per group, per sex.

Male animals treated in utero with Dex but reared by Veh-treated mothers showed a blunted corticosterone peak response (Fig. 5A) and low corticosterone levels by 60 min compared with the control group (Veh-Veh). Similarly, the female Dex-Veh group had low corticosterone levels at 60 min compared with Veh-Veh females (Fig. 5B). In contrast, animals reared by Dex-treated mothers (Veh-Dex and Dex-Dex) had a significantly prolonged adrenocortical response compared with Veh-Veh animals. Male Veh-Dex and Dex-Dex male animals had significantly elevated levels of corticosterone at 30 and 60 min, whereas females exhibited elevated levels at 60 min. In addition, Dex-Dex females failed to return to baseline corticosterone levels 120 min after the restraint, indicating a greater failure to "shut down" the adrenocortical response to stress compared with all other female groups.

Corticoid Receptors in the Hippocampus

Comparison of the effect of prenatal Dex treatment across gender is possible, because both male and female sections were processed simultaneously in each of the in situ experiments and the autoradiograms had equal representation of all groups. The GR mRNA densitometric analysis followed by a three-way ANOVA revealed significant sex (F = 29, P = 0.01), treatment (F = 11.3, P = 0.01), and hippocampal region effects (F = 255, P = 0.0001). In addition, when a three-way ANOVA was performed by hippocampal region using sex, rearing mother, and in utero exposure as factors, a consistent mother and fetal exposure interaction was identified for CA3-4 (F = 7, P = 0.009) and DG (F = 9, P = 0.005). A sex effect was also found over the CA2 (F = 40, P = 0.0001) and CA3-4 regions (F = 31, P = 0.0001).

Post hoc analysis revealed that overall, females exhibited a significantly lower GR mRNA intensity over the CA3-4 and CA2 regions compared with males (~20-40%, respectively, see Figs. 6 and 7, A and B). Specific and consistent regional effects on GR gene expression were detected in the hippocampus of both males and females subjected to different in utero and rearing conditions. Specifically, compared with Veh-Veh animals, the Dex-Veh groups had ~20% increase in the CA1 and DG region. Dex-Veh males also showed ~60% increase in the CA3-4 subfield (Veh-Veh = 18 ± 2; Dex-Veh = 29 ± 1; mean gray level ± SE, P < 0.001). In contrast, Dex-Dex animals had an ~20% decrease of hippocampal GR mRNA expression over the DG region (Dex-Dex, males = 35 ± 1; females = 34 ± 3; P < 0.05). With the exception of a sex effect (F = 42, P = 0.0001), hippocampal MR mRNA quantification did not reveal any statistically significant differences among groups (data not shown, see Fig. 8 for photo).


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  		Fig. 6.   Representative photomicrograph of the glucocorticoid receptor (GR) mRNA in animals exposed to Dex or Veh in utero. Because animals were cross-fostered at birth, the following groups resulted: 1) Veh-Veh (offspring exposed to Veh in utero, rearing mother treated with Veh during gestation), 2) Veh-Dex (offspring exposed to Veh in utero, rearing mother treated with Dex during gestation), 3) Dex-Veh (offspring exposed to Dex in utero, rearing mother treated with Veh during gestation), and 4) Dex-Dex (offspring exposed to Dex in utero, rearing mother treated with Dex during gestation), n = 6-8 per group, per sex. CA, cytoarchitectural; DG, dentate gyrus.



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  		Fig. 7.   Densitometric analyses of GR mRNA expression through the different subfields of the hippocampal formation in males (A) and females (B). The 4 different rearing and in utero treatment groups are shown. n = 6-8 animals, per group, per sex. Significant sex, treatment, and hippocampal region effects were detected by ANOVA. *P < 0.05 vs. Veh-Veh, means ± SE, n = 6-8 per group, per sex.



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  		Fig. 8.   Representative photomicrograph of the mineralocorticoid receptor (MR) mRNA in animals exposed to Dex or Veh in utero. A: males; B: females. With the exception of a sex effect (F = 42, P = 0.0001), hippocampal GR mRNA quantification did not reveal any statistical significant differences among groups.

We also quantified GR mRNA expression in the paraventricular nucleus of the hypothalamus. No statistically significant differences were found among groups. However, a two-way ANOVA revealed a sex effect (F = 4.4, P = 0.04). Consistently, females had lower GR mRNA levels compared with the male animals, but this was most pronounced in the Dex-Dex group (Veh-Veh: males = 46.4 ± 8.4, females = 40.2 ± 2.9; Dex-Veh: males = 45.1 ± 4.6, females = 43.1 ± 1.4; Veh-Dex: males = 46.3 ± 2.8, females = 44.4 ± 4.1; Dex-Dex: males = 53.3 ± 4.2, females = 37.3 ± 0.7, means ± SE).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study investigated the effect of prenatal Dex exposure on early perinatal events. We found that exposure to Dex at a dose of 0.27 mg · kg-1 · day-1 from GDs 15 to 20 results in maternal suppression of the hypothalamic-pituitary-adrenal axis and causes increased mortality of the offspring during the perinatal period. Low birth weights, poor weight gain, and restless behavior in the mothers during the first week of postpartum were also observed. Effects on hippocampal function are complex and were influenced by postnatal maternal factors. As adults, the offspring that received Dex in utero and were reared by mothers that also received Dex during gestation had impaired performance in the water maze spatial task. These animals also had inability to rapidly terminate the adrenocortical response to acute restraint stress and decreased hippocampal GR mRNA expression. In contrast, Dex-exposed animals reared by Veh-treated mothers have adequate performance in the water maze spatial task, showed reduced adrenocortical response to acute stress, enhanced glucocorticoid feedback sensitivity, and increased hippocampal GR gene expression. These findings on the Dex-Veh offspring were completely unexpected and point to the long-lasting influence of postnatal maternal factors in shaping brain development and function. Veh-treated mothers, in effect, prevented the adverse effects produced by prenatal Dex exposure on cognitive and stress responsiveness.

Our study suggests that chronic exposure to Dex in utero has an impact on perinatal survival and somatic growth. This is in agreement with other investigators who performed prenatal Dex treatment with similar doses as we used in our study. For example, Eishi and co-workers (15) reported two different postnatal offspring patterns of survival when pregnant mice were administered a similar Dex dose as we did in our studies (0.2 mg · kg-1 · day-1 from GDs 15 to 20). A wasted appearance and death within 1 week of life characterized one group of survivors. The remaining pups showed long-lasting growth retardation. Long-lasting impairments of body growth were also described in the offspring of rats treated with Dex (0.2 mg · kg-1 · day-1) early in pregnancy (6, 40). In our study, the lack of adequate weight gain observed in Dex-treated mothers during the latter part of gestation reflects the detrimental effects of Dex to the mother and fetus during pregnancy and is likely to be contributing to the immediate and long-term effects observed in the offspring. Previous studies did not monitor mother's weight gain during pregnancy and during postpartum period or explore the possibility that maternal care or the quality and quantity of milk production were significant contributing factors in the long-lasting growth impairments. We limited the intrusiveness of our physical and behavioral measures to avoid disturbing the infant-maternal interactions. We observed that Dex-treated mothers spent significantly more time eating and drinking during the second week postpartum compared with the control mothers. Thus it is possible that the catch-up weight gain during the postnatal period, which would certainly be beneficial for the mother, may have deviated the overall maternal care given by the Dex-treated mothers to their young. Interestingly, animals exposed to Veh in utero but reared by Dex-treated mothers did not have growth problems. This suggests that pups exposed to Dex in utero were intrinsically at a disadvantage at birth and were most affected by postnatal maternal events. Taken together, the present study indicates that chronic exposure to Dex in utero has an impact on the maternal behavior, the somatic growth of the developing fetus, the immediate natal outcome of the newborn, and the postnatal growth and development of the offspring.

Our data also suggest that in utero Dex treatment has an impact on hippocampal biological and functional measures and that maternal factors can modify these effects. Animals reared by Dex-treated mothers displayed a small, but significantly decreased, learning ability in the Morris water maze that correlated with a prolonged adrenal response to acute stress. In contrast, those animals exposed in utero to Dex but reared by Veh-treated mothers had adequate learning of the spatial task and a swift termination of the stress response. It is evident from a number of studies that acquisition and memory in spatial avoidance tasks are closely linked to the quality of the stress response. The consensus is that these capacities are interrelated (8, 10). Stress hormones, in particular, corticosterone, are released during learning and are necessary for establishment of enduring memory. Corticosterone facilitates spatial memory when given immediately after the task in amounts comparable to a peak stress response (39). On the other hand, GR antagonism impairs this effect (34). Spatial learning is also impaired if an acute stressor is introduced during the training session giving rise to a prolonged corticosterone exposure (12). Thus cognitive functions are impaired both in the absence of appropriate GR activation (23) and/or if the activation is sustained (39). Our data are in agreement with these notions.

In the present study, we found that the pattern of hippocampal GR gene expression closely paralleled a pattern that would be predicted from the stress response and visual-spatial learning curve. A high GR expression correlated with a swift termination of the stress response in animals exposed in utero to Dex but reared by Veh-treated mothers, whereas low glucocorticoid expression was associated with an impaired "shut down" in in utero-treated Dex animals reared by Dex-treated mothers. Two possible explanations for this pattern of GR mRNA expression come to mind. First, there is evidence to support that mother-pup interactions may influence the offspring's long-term GR expression, behavioral outcome, and quality of the stress response (25, 26, 41). Specifically, increased GR expression in the hippocampus and reduced adrenocortical responses to stress are associated with brief periods of daily handling for the first weeks of life. These effects have been associated with decreases in body temperature, which activate thyroid hormone release and mediate increased hippocampal GR expression (30). Although probably mediated by different mechanisms, the same is also observed in the offspring of mothers that actively groom their pups during the first 10 days of life (26). The dam spends more time giving nutrition, stimulation, and warmth to a litter that is perceived to have poor health (7, 17, 20, 47). However, in our study, we did not observe a difference in the time spent in the nest, in active nursing, or grooming by the Veh or Dex suckling dam. Probably this is due to the fact that the dams had a mixed population of prenatally treated Dex and Veh pups. It has been observed that in litters with pups of different nutritional status, the time that the dam spends nursing and grooming is not significantly increased compared with litters with homogeneous healthy pups (20). Alternatively, it is possible that in our study, all pups were perceived to be healthy by the mothers after day 3 of life when infant deaths ceased. Yet, it is evident from our video recording that Dex-treated mothers behaved different from the Veh controls. Dex-treated mothers had significantly increased activity outside the nest and fewer resting periods compared with Veh mothers, particularly after the first week of lactation. This observation coincides with an increase of self-grooming and eating behavior concomitant with a catch-up weight gain in the Dex-treated mothers. Recall that we controlled for the composition of the litters to include both pups exposed to Dex prenatally and pups that were not. Thus this increased time outside the nest and reduced rest periods appear to be linked to maternal self-directed behaviors, but ultimately time spent outside the nest and reduced rest periods appear to be important factors influencing differentially the long-term repercussions in hippocampal gene expression and function of the offspring. Interestingly, the altered biological measures were most extreme in the offspring reared by Dex-treated mothers that also had the in utero Dex exposure. These effects are to some extent also seen in pups that were treated in utero with vehicle but reared by Dex-treated mothers. What is remarkable is the fact that the effects on learning, quality of the stress response, and GR expression in the hippocampus were prevented when animals exposed to Dex in utero were reared by vehicle-treated mothers.

It is possible that exposure to glucocorticoids during early postnatal life, in addition to the prenatal exposure, also played a role on the biological and behavior measures. Glucocorticoids, both endogenous and fluorinated, are secreted in breast milk (35). Therefore, we cannot exclude the possibility that pups reared by in utero Dex-treated mothers were exposed to small, although nevertheless potent, amounts of a GR-binding agent during the first days of lactation. Dex exposure during days 1, 3, and 5 of lactation (1 µg/g sc) has been shown to selectively affect GR expression in the hippocampus of adult offspring (16). Considering the likelihood of exposure to Dex through breast milk, our study may reflect that selective occupation of hippocampal GR both prenatally in utero and postnatally. The latter combination may contribute to express a different functional phenotype compared with that which results from selective postnatal life occupation of GR. A functional phenotype is theoretically possible because exposure to Dex during prenatal or both pre- and postnatal development can alter patterns of GR abundance and distribution that are unique to the fetal and postnatal periods (27, 33, 39). It is tempting to speculate that selective activation and subsequent downregulation of particular hippocampal neurons expressing GR during fetal versus postnatal period altered the developmental program and final expression in the adult hippocampus. Another possibility is that the relative absence of circulating corticosterone, as a result of adrenal suppression during these periods, has detrimental effects on the development of brain areas that contribute to the functions that we tested (11).

Beyond the corticoid receptor effects identified in the hippocampal formation one would also have to consider more complex Dex effects at the cellular structural level that would have repercussions on the basic circuitry "blueprint" in the developing brain. Neuronal maturation, replication, differentiation, planned cell death, and the organization of synaptic connections between cells is affected by Dex exposure (32). In particular, a reduced number of dendritic spines and synaptic contacts are observed in cortical layers and in the hippocampus interneurons as a consequence of prenatal glucocorticoid exposure (33, 43). Decreased myelination is also readily observed (21), and long-lasting deficits that result from Dex exposure appear to be restricted to regions undergoing rapid mitosis during the period of drug exposure. Of importance to our learning and memory tasks is the work of Carlos and colleagues (6). This group showed that Dex given at the same doses as in this study caused an unfolding of developmental events that led to permanent neuronal deficit in the forebrain area and in terminal projections from forebrain into prefrontal cortex. Numerous glial cells substitute the forebrain and cortical neuronal layers. Detailed morphology was not performed in this study, but the authors speculate that axonal projections were likely to be affected as well. Detailed morphology was not performed in our study either, but the possibility exists that projections within the hippocampus and those arising from the hippocampus onto prefrontal cortical areas, which are involved in spatial working memory, may have been permanently affected in the Dex-exposed animals reared by Dex mothers. Similarly, other areas linked with primary sensory integration, working, and executive memory may have been adversely affected. However, our data suggest that regardless of the process or processes involved, the rearing of Veh mothers mitigates some of these generalized Dex effects.

In summary, the developing LHPA axis is sensitive to low-dose in utero Dex exposure during early gestation. Long-term detrimental effects on learning and quality of the stress response are observed. These effects are more pronounced in males than in females. However, a control mother providing a different rearing environment than those mothers treated with Dex during pregnancy can influence positively and permanently the expression of key molecules involved in the quality of the stress response and cognitive function.

Perspectives

Dex treatment is readily used prenatally and immediately before birth in humans. Although some medical conditions that require this treatment are rare, others are very common. Congenital adrenal hyperplasia (CAH) is an example of a relatively rare condition, but one that requires Dex treatment as early in the first trimester of pregnancy as possible. A more common use of Dex is its administration to pregnant women to mature the fetal lungs of very premature fetuses at risk for early delivery. Clearly Dex prevents virilization of a female fetus affected with CAH and causes fetal lungs to rapidly mature in premature fetuses. However, Dex also has the potential of affecting negatively the rapidly developing brain. The present study fills an important gap in the literature with regard to the combined effect of steroids on the brain of offspring exposed to Dex in utero, as well as the behavior, of the mother through which the fetus receives Dex treatment. The importance of the role of postnatal care is the most important aspect of these findings. The fact that healthy mothers that did not receive Dex prevented the adverse brain-cognitive effects of the Dex-exposed pups is a finding that, if replicated in human studies, can have potentially important ramifications for preventive care of the premature infant and other infants requiring early life Dex treatment. Our findings also point to greater cognitive consequences to the male fetus, probably due to the Dex suppression of fetal testosterone biosynthesis (24) and placental estradiol biosynthesis, hormones that have been implicated in the superior abilities for spatial learning in male rats. Although it is unclear whether these findings are directly transferable to the human condition, we believe that there is a need for clinical studies that examine the long-term cognitive effects of Dex treatment during the prenatal period, especially in male offspring.


    ACKNOWLEDGEMENTS

The authors to thank Ramin Eskandari for technical assistance.


    FOOTNOTES

This work was supported by National Institute of Drug and Addiction Grant DA-00250 and National Institute of Mental Health Grant MH-42251.

Address for reprint requests and other correspondence: D. M. Vázquez, 8240 Medical Science Research Bldg. III, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0646 (E-mail: dmvazq{at}umich.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 16 December 1999; accepted in final form 26 July 2000.


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DISCUSSION
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