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Prenatal psychological stress causes higher emotionality, depression-like behavior, and elevated activity in the hypothalamo-pituitary-adrenal axis
Neuroscience Research 59 (2007) 145–151 www.elsevier.com/locate/neures
Prenatal psychological stress causes higher emotionality, depression-like behavior, and elevated activity in the hypothalamo-pituitary-adrenal axis Hiroshi Abe a,*, Noriko Hidaka a, Chika Kawagoe a, Kei Odagiri a, Yuko Watanabe a, Testuya Ikeda b, Yuta Ishizuka a, Hiroyuki Hashiguchi a, Ryuichiro Takeda a, Toshikazu Nishimori b, Yasushi Ishida a a
Division of Psychiatry, Department of Clinical Neuroscience, School of Medicine, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan b Division of Neurobiology, Department of Anatomy, School of Medicine, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan Received 21 February 2007; accepted 14 June 2007 Available online 19 June 2007
Abstract In humans, stressful events during pregnancy may raise the risk of psychiatric disorders in offspring, and studies with rodents have found that physical prenatal stress can cause changes in the physiology, neurobiology, and behavior of offspring. In the present study, we examined whether psychological prenatal stress with little physical stress could cause changes in the neurobiology and behavior of offspring in Sprague–Dawley rats, as physical prenatal stress did. Dams received psychological stress by observing a rat being electrically shocked behind a transparent wall in the social communication box during the last trimester of gestation but were not exposed to any physical stress. Male offspring from the dams exposed to psychological stress showed enhanced emotionality in an open field test, depression-like behavior in a forced swim test, and enhanced activity in the hypothalamo-pituitary-adrenal axis, compared with rats from untreated dams. However, the prenatally stressed rats showed intact ability to acquire context conditioning. This is the first report that psychological prenatal stress in the communication box can cause changes in the neurobiology and behavior of offspring in rodents. # 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Prenatal psychological stress; Anxiety; Depression; Fos immunohistochemistry; Hypothalamic-pituitary-adrenal axis; Communication box
1. Introduction In humans, it has been reported that stressful events during pregnancy may raise the risk of psychiatric disorders in offspring (Wadhwa et al., 2001; Weinstock, 2001; Maccari et al., 2003). Studies with rodents give us a clearer relationship between stressful treatment during pregnancy and changes in the physiology and behavior of the offspring (Kofman, 2002; Maccari et al., 2003; Weinstock, 2005). Generally, prenatally stressed rats showed higher emotional reactivity, higher levels of anxiety, and depression-like behavior. In such studies, physical stress was given to the dams. For example, restraint stress, to which dams were exposed for 45 min three times per day with bright light during the last trimester of gestation, * Corresponding author. Tel.: +81 985 85 2969; fax: +81 985 85 5475. E-mail address: [email protected] (H. Abe).
has been most widely used in recent studies with rodents (Bhatnagar et al., 2005; Viltart et al., 2006; Wang et al., 2006). Additionally, foot shock (Estanislau and Morato, 2005), injection of saline (White and Birkle, 2001), loud noise (Wakshlak and Weinstock, 1990), or a combination of these (Koenig et al., 2005) were used as physical stress. However, in humans, we may say that pregnant women are exposed to psychological stress more frequently than to physical stress. Although pregnant women may experience physical injury caused by accidents or infections, they are more often exposed to psychological stress, such as anxiety about their babies and their entirely new lifestyle, financial problems, workplace worries, and so on. Therefore, to examine the etiology of psychiatric disorders caused by prenatal stress in humans, one should use an animal model, in which the effects induced by psychological stress but not physical stress can be evaluated. However, as stated above, to our knowledge, physical stress was
0168-0102/$ – see front matter # 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2007.06.1465
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given to the dams in almost all studies with rodents. In fact, Chapillon et al. (2002) reviewed a series of their studies with emotional stress. In their experiment, where they exposed dams to a cat as a prenatal stress, exposure to a cat was acutely carried out at gestational days 10, 14, or 19. On the other hand, we intended to expose dams to psychological stress during the last trimester of gestation to compare directly the effect of conventional restrain stress procedure (Bhatnagar et al., 2005; Viltart et al., 2006; Wang et al., 2006). Furthermore, according to their description (Lordi et al., 1997), when dams were put in a box with a cat together, ‘‘rats exhibited strong motor and autonomic reactions’’. This suggested that dams and fetus would have considerable physical stress as well as psychological stress. Moreover, intensity of stress would be difficult to control because a cat put in a box would not behave against each pregnant rat in a similar manner. Therefore, in this study, we used psychological stress produced by the social communication box (Ogawa and Kuwabara, 1966). The communication box is an apparatus in which subjects observe a rat being electrically shocked behind a transparent wall. This seems to expose subjects to psychological stress with no physical stress. To examine emotional reactivity, depression-like behavior, and learning ability of male offspring from the dams exposed to psychological stress, comparing with offspring from untreated dams, rats were tested in an open field test, a forced swim test, and a context conditioning. Additionally, neurobiological differences were examined by measuring plasma corticosterone contents and the number of Fos immunopositive cells in some brain regions between them. 2. Materials and methods 2.1. Animals Six Sprague–Dawley pregnant rats (9-week-old) at the 13th gestational day and six Sprague–Dawley female rats (10-week-old) were obtained from Charles-River, Japan, and were individually housed on a 12 h light/dark cycle (light on: 08:00 h) with free access to food and water. The pregnant rats were nulliparous until this pregnancy. The pregnancy was confirmed by the existence of the plug in the vaginas. Three pregnant females were given psychological stress and the others were not. Six nonpregnant female rats were used as ‘‘presenters’’ that actually received the electrical foot shock. The ethical committee for animal experimentation at University of Miyazaki approved the experimental protocols used.
2.2. Prenatal stress procedure A communication box with a grid floor composed of 5 mm diameter stainless steel rods placed 13 mm apart was used. It had four compartments (15 cm 15 cm) divided by transparent plastic walls. An electric shock generator (MSG-001, Toyo Sangyo, Japan) was controlled by a program of our own making. Electric foot shocks (0.3, 0.4, or 0.5 mA) for 1 s were randomly presented once every 60 s. There were plastic plates on the grid floor in three compartments and pregnant rats put in these compartments never had electric shocks, while the ‘‘presenter’’ rat was put in the compartment without a plastic plate. Three sessions for stress exposure were carried out for 60 min at 09:00, 12:00, and 14:00 h in 1 day. These stress sessions were carried out during gestational day 13–20. The presenter rat was changed every session, so each presenter rat was used every six sessions. All six pregnant females were transported to a waiting room from home cages at an animal center every day and handled to weigh their body weight. Three pregnant rats were given psychological stress in above procedure after transferring from the waiting room to an experiment room. The other three dams were kept in the waiting
room, and they were not given psychological stress. The pups were weaned at 21 days of age and housed in groups of three or four in a cage. Only male rats were used in this study. Offspring from the dams exposed to psychological stress were used as the prenatally, psychologically stressed (PSY) group, and offspring from the dams without any treatment were used as a control (C) group. All rats performed open field test, but different animals were used in other behavioral tasks to suppress the confounding factors to the minimum, because discomfort experience in the context conditioning might affect the performances in the forced swim test carried out in the same laboratory.
2.3. Open field test All rats in groups C (n = 20) and PSY (n = 20) were tested in an open field arena at postnatal day (PND) 60. The open field apparatus was a square box (100 cm 100 cm 40 cm) constructed of gray Plexiglas. Testing was conducted under dim light (about 80 lx). The rat’s behavior was recorded using a video camera and analyzed from the videotapes, using the motion tracking system (Dipp-Motion 2D, DITECT, Japan). The rat was placed in the center of the arena and behaved freely for 5 min. The numbers and durations of the following behavioral parameters were recorded during 5 min: rearing, standing with the body inclined vertically, forequarters raised with and without its forelimb touching the wall of the apparatus; the total time spent in the center square (33 cm 33 cm); the total time spent in the four corners (12 cm 12 cm at each corner); traveling speed and total distance traveled; exploration, at-least-once entered open field units, that is, the area covered by rats during a 5 min period. Scores ranged from 0 to 9. The number of rearings was manually counted by experimenters, and other indexes were analyzed with the above motion tracking system.
2.4. Context conditioning Rats in groups C (n = 6) and PSY (n = 6) were tested in the context conditioning at PND 110. They were tested in the open filed test at PND 60, and they came from all six litters (two rats per litter) The testing apparatus was a shock box (29 cm 26 cm 28 cm) with a stainless-steel bar floor placed in an isolation chamber with a semitransparent window to allow behavioral observation. A mild foot shock (0.3 mA; 60 Hz) was generated with a shock generator using a scrambler (Toyo Sangyo Co., Toyama, Japan) and delivered through the grid chamber floor. The timing of the shocks was controlled with a repeated-cycle timer; they lasted 1 s and were delivered every 15 s for 20 min. Rats were re-exposed to the testing apparatus in which they had received the foot shock treatment 1 day before. The duration of immobile behavior during 10 min and the latency until the first movement were recorded. The duration of immobility, defined as the animal being motionless but alert, was measured by directly observing the motion of each animal during the 10 min period in the testing apparatus in the PSY and C groups.
2.5. Forced swim test A modified version of the forced swim test (Porsolt et al., 1977, 1978) was performed by rats in groups C (n = 6) and PSY (n = 6) at PND 120. They came from all six litters (two rats per litter) and tested in the open field test at PND 60, but did not experience the context conditioning at PND 110. A water tank (height = 35 cm; diameter = 30 cm) was filled with 20 8C water up to a level of 22 cm. In the first day, rats were placed in the water tank for 15 min. The water was changed for every rat. Rats were again placed in the water tank for 5 min on the next day. The duration of immobility behavior (floating in water without active movements of forepaws) was measured by experimenters.
2.6. Immunohistochemical analysis Rats in groups C (n = 6) and PSY (n = 5) which performed the open field test at PND 60 but did not experience context conditioning or the forced swim test were used. They were deeply anesthetized at PND 130 with sodium pentobarbital and perfused transcardially, first with heparinized saline, then with 500 ml of cold, freshly prepared 4% paraformaldehyde, and finally with 500 ml of 10% sucrose in 0.1 M phosphate buffer. The brains were removed immediately, immersed in 30% sucrose in 0.1 M phosphate buffer for 2 days at 4 8C,
H. Abe et al. / Neuroscience Research 59 (2007) 145–151 and then cut coronally into 50 mm sections on a freezing microtome for immunohistochemical examination. From each animal section through the prefrontal cortex (PFC; approximately 3.2 mm rostral to the bregma), the hypothalamic paraventricular nucleus (PVN; approximately 1.8 mm caudal to the bregma), the amygdala (AMY; approximately 2.8 mm caudal to the bregma), the dorsal raphe nucleus (DR; approximately 7.8 mm caudal to the bregma), and the locus coeruleus (LC; approximately 10.0 mm caudal to the bregma) were selected for histological study (Paxinos and Watson, 1998). The sections were collected in a phosphate-buffered saline solution (PBS; pH 7.4) and processed for Fos immunohistochemistry according to the manufacturer’s instructions for use with the streptavidin–biotin system (Histofine SAB-PO (R) kit, Nichirei, Tokyo, Japan). After incubation in 10% normal goat serum for 20 min, the sections were incubated at 4 8C overnight with a Fos antibody, a rabbit polyclonal antibody raised against a peptide corresponding to human c-fos amino acid residues 3–16 (diluted 1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). They were then rinsed three times in PBS and incubated at room temperature for 45 min with a secondary biotinylated goat antirabbit IgG, again rinsed three times in PBS, and further incubated at room temperature for 15 min with a streptavidin–peroxidase complex. After three rinses in PBS, the reaction products of biotinylated goat antirabbit IgG and streptavidin-conjugated horseradish peroxidase were intensified by pretreatment with 0.125% cobalt chloride, then visualized using 0.01% diaminobenzidine tetrahydrochloride and 0.0003% hydrogen peroxide. The sections were mounted on gelatin-coated glass slides, air-dried, dehydrated, cover-slipped, and Fos immunoreactivity was analyzed using light microscopy. The two most heavily labeled sections through each structure of each animal were chosen for cell counting. The number of Fos immunopositive cells was counted in two sections per structure in each animal in a standardized manner under 200 magnification using a microscopic 0.25 mm 0.25 mm grid. The grid was placed in the center of the eye piece. The average number of Fos immunopositive cells in each structure was computed.
2.7. Plasma corticosterone assay A blood sample (1 ml) was collected from the right atrium under deep anesthesia before the perfusion described above. Heparin (10 ml) was mixed into a sample of collected blood. This sample was centrifuged at 6100 g at 4 8C for 25 min, and the supernatant was diluted with a buffer supplied by the manufacturer. Plasma corticosterone content was measured by ELISA using a commercially available kit (rodent corticosterone ELISA test kit, Endocrine Technologies Inc., USA). ELISA was performed in a 96-well plate, where antibodies were coated. Standards and samples were pipetted into appropriate wells, and a solution of corticosterone labeled with horse radish peroxidase was added to each well. After incubation for 2 h at 37 8C, the reaction was terminated by washing the plate with wash solution. Colorimetric detection of peroxidase activity was achieved by adding tetramethylbenzidine solution and incubating for 20 min at room temperature. The enzymatic reaction was stopped with stopping solution, and the optical density of each well was measured at 450 nm using a plate reader (MTP-500, Corona Electric Co. Ltd, Japan). A standard curve was produced by using six standard values, in which the absorbency values for blank tubes were subtracted. Results for the samples were read from this standard curve by a computer program.
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exposed to stress and dams not exposed to stress were 305.8 15.6 and 309.1 14.8, respectively. On the last day for the stress exposure, they were 349.8 16.2 and 358.0 17.9, respectively. That is, gains of body weights of dams exposed to psychological stress were slightly lower than dams without stress exposure. However, the differences were not significant. Body weights of offspring were analyzed at PND 60 and 105. There were no significant difference in the body weights between PSY (278.3 3.7) and C (270.3 5.0) at PND 60 [F(1,38) = 1.67, p = 0.20]. At PND 105, the body weights of PSY (460.0 5.8) tended to be greater than C (437.4 10.1) [F(1,37) = 3.9, p < 0.1]. One rat of C group was found dead in the morning at PND 61, and cause of the death was uncertain. 3.2. Open field test There was no significant difference in the total time spent in the center area between PSY and C [F(1,38) = 1.14, p = 0.29] (Fig. 1A). On the other hand, rats in group PSY spent significantly more time in the corners of the arena than those in C [F(1,38) = 6.74, p < 0.05] (Fig. 1B). PSY showed significantly less distance traveled during the 5 min session than C [F(1,38) = 5.98, p < 0.05] (Fig. 1C). Furthermore, the moving speed was also lower in PSY than in C [F(1,38) = 4.46, p < 0.05] (Fig. 1D). The number of rearings in PSY was significantly less than in C [F(1,38) = 4.32, p < 0.05] (Fig. 1E). PSY explored a significantly lower number of divisions in the open field arena than C [F(1,38) = 10.06, p < 0.005] (Fig. 1F).
2.8. Statistical analysis One-way ANOVAs were used to assess the differences among measurements in the open field test, context conditioning, and forced swim test. The body weights, the number of Fos immunopositive cells in each brain area, and the level of plasma corticosterone in the C and PSY groups were analyzed by one-way ANOVA.
3. Results 3.1. Body weights of dams and offspring Body weights of dams during stress exposure were analyzed. On the initial day, mean body weights standard error of dams
Fig. 1. Effects of prenatal psychological stress on the open field test. (A) Time spent in the center square (33 cm 33 cm) and (B) in the corners (12 cm 12 cm for each corner). (C) Total distance traveled in the arena. (D) Moving speed. (E) Number of rearings. (F) Number of divisions explored. Data are expressed as means + S.E.M. **p < 0.005, *p < 0.05.
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H. Abe et al. / Neuroscience Research 59 (2007) 145–151 Table 1 Number of Fos immunopositive cells in five brain areas of C and PSY Area
PFC PVN AMY DR LC
Treatment C
PSY
4.9 2.8 17.9 4.1 10.3 3.5 9.2 3.6 21.9 6.0
18.3 8.9 34.3 3.6* 7.1 1.7 23.6 4.1* 38.6 9.6
Value represents the mean number S.E.M. PFC: prefrontal cortex, PVN: hypothalamic paraventricular nucleus, AMY: amygdala, DR: dorsal raphe nucleus, and LC: locus coeruleus. Significant differences between PSY and C were detected in PVN and DR (*p < 0.05).
LC: F(1,9) = 2.34, p = 0.16]. In PVN (Fig. 4) and DR, the number of Fos immunopositive cells was significantly higher in PSY than in C [PVN: F(1,9) = 8.56, p < 0.05; DR: F(1,9) = 7.18, p < 0.05]. Fig. 2. Effects of prenatal psychological stress on the context conditioning. (A) Latency of the first movement and (B) duration of immobility behavior in the shock box. Data are expressed as means + S.E.M. No significant difference was detected.
3.3. Context conditioning The duration of immobility during 10 min (Fig. 2A) and the latency of the initial movement (Fig. 2B) were analyzed. There were no significant differences between these measurements for PSY and C [F(1,10) = 0.99, p = 0.34, F(1,10) = 2.79, p = 0.13, respectively]. 3.4. Forced swim test In the forced swim test, the duration of immobility during the 5 min test session was significantly longer in PSY than in C [F(1,10) = 6.61, p < 0.05] (Fig. 3). 3.5. Immunohistochemistry (Table 1) In PFC, AMY, and LC, there was no significant difference in the number of Fos immunopositive cells between PSY and C [PFC: F(1,9) = 2.41, p = 0.15; AMY: F(1,9) = 0.61, p = 0.45;
Fig. 3. Effects of prenatal psychological stress in the forced swim test. Duration of immobility behavior in test session. Data are expressed as means + S.E.M. *p < 0.05.
Fig. 4. High-magnification, bright-field photomicrographs showing the enhancement of Fos immunopositive cells in the paraventricular hypothalamic nucleus (PVN) of C and PSY groups. The square indicates the region in which the number of Fos immunopositive cells was counted. 3V: third ventricle. Scale bar = 100 mm.
H. Abe et al. / Neuroscience Research 59 (2007) 145–151 Table 2 Basal plasma corticosterone in PSY and C Treatment
Plasma corticosterone content
C PSY
89.3 24.6 172.0 18.1*
Value represents the mean concentration S.E.M. (ng/ml). The basal level of plasma corticosterone of PSY was significantly higher than that of C (*p < 0.05).
3.6. Plasma corticosterone assay (Table 2) The basal level of plasma corticosterone was significantly higher in PSY than in C [F(1,9) = 6.70, p < 0.05]. 4. Discussion In the present study, we demonstrated that psychological stress given to pregnant dams could cause enhanced emotionality, depression-like behavior, and enhanced activity in the hypothalamo–pituitary–adrenal (HPA) system of offspring in rats. In this study, the dams delivering C groups were not placed in the novel communication box, while the dams delivering PSY groups were placed in such a box. Therefore, exposure to the novel environment, in addition to the exposure to shocked rats, may produce the effects between two groups. However, in any case, exposed stress mainly involved much psychological factor and less physical factor, comparing restraint stress (Bhatnagar et al., 2005; Viltart et al., 2006; Wang et al., 2006), which is most widely used as prenatal stress procedure. The gains of body weights of dams exposed to psychological stress were slightly lower than dams without stress exposure. However, the differences were not significant. Some researches have reported that prenatal stress reduce body weight of offspring (Herrenkohl, 1979; Rhees and Fleming, 1981; Pollard, 1984), however such reduction of body weight was not found at PND 60 and 105. Furthermore, any prenatally stressed rats did not die through this experiment. In these respects, the effect of psychological stress exposed to the dams in this experiment would be milder than the stress provided by conventional procedures. In an open field test, rats are exposed to contradictory motivations to avoid aversive stimuli (novelty, lit, and open area) and to explore new places (Crusio, 2001). The internal conflict is assumed from some behavioral measurements. For example, the wall-seeking behavior, which is measured by duration to stay the peripheral area near the wall and to avoid the center area in the present study, is called thigmotaxis, and more thigmotactic behavior is considered to reflect more emotionality (anxiety or fear) because this behavior may reflect phylogenetically prepared fear reactions to avoid predators (Treit and Fundytus, 1989). This thigmotaxis has been used to measure fear or anxiety in open field tests. On the other hand, the distance traveled, the number of rearings, and the number of at-least-once-entered open field units has been thought to reflect animals’ exploratory tendencies. In the present study, there was no significant difference in the duration spent in the center
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area between the C and PSY groups, while PSY showed significantly more time spent in the corners, less distance traveled, fewer rearings, and fewer at-least-once-entered open field units than C. These results suggest that PSY should show higher emotionality and lower tendency to explore than C. Elevated emotionality has been reported in findings using prenatally, physically stressed rats in an open field test (Wakshlak and Weinstock, 1990; Poltyrev et al., 1996; Van den Hove et al., 2005). Furthermore, enhanced emotionality of prenatally stressed rats has been also reported in the elevated plus maze task (Wakshlak and Weinstock, 1990), a task often used to examine an animal’s fear or anxiety (Pellow and File, 1986). The present result suggests that even psychological stress with little physical stress to pregnant dams could change emotionality in offspring. In the context conditioning, the ability of rats to learn and remember an association between an aversive stimulus and contextual cues can be examined (Fanselow, 1980). There were no significant differences in the latency of initial movement and the duration of freezing between the C and PSY groups. Some researchers reported that severe prenatal physical stress disrupted this conditioning (Fride et al., 1986; Shiota and Kayamura, 1989), while mild stress enhanced the conditioning (Fujioka et al., 2001). On the other hand, the present results suggest that rats whose mother was exposed to psychological stress will not have deficits in learning and memory about an association between an aversive stimuli and context. Additionally, the number of Fos immunopositive cells in the amygdala, which is considered to be closely involved with context conditioning (Fanselow and Gale, 2003), did not differ significantly between PSY and C. Thus, the prenatal psychological stress in this study may not have been strong enough to cause any behavioral and neurobiological changes in the conditioning. In the forced swim test, the time spent swimming and the time spent floating in a cylinder filled with water were measured (Porsolt et al., 1977, 1978). The present results indicate that rats become more depressed because immobility increased and struggling behavior decreased. The validity of this test is recognized to some extent because the immobility behavior is decreased by many types of antidepressants and increased by stressors including immobilization, foot shock, and intruder encounters (Weiss et al., 1981; Zebrowska-Lupina et al., 1990; Hebert et al., 1998). Some studies following physical prenatal stress (Morley-Fletcher et al., 2003, 2004) demonstrated increased immobility behavior, suggesting that prenatally stressed rats will be more depressed than normal rats. The results of the present study suggest that prenatal stress given psychologically with little physical stress could make rats more depressed than untreated rats. Serotonergic neurons in the dorsal raphe nucleus are considered to play important roles in depression (Bermack and Debonnel, 2005). In the present study, the number of Fos immunopositive cells in DR of PSY was significantly higher than that in C. This suggests that depression-like behavior shown in PSY was related to the enhanced activity of serotonergic neurons of DR. Prenatally stressed rats, compared with normal rats, usually showed elevated level of plasma corticosterone (Weinstock
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et al., 1998; Maccari et al., 2003). For example, Weinstock et al. (1998) found a significantly elevated level of corticosterone at the baseline (before foot shock) and 15 min after foot shock in rats with prenatal stress. Correspondingly, in the present study, the basal level of plasma corticosterone was significantly higher in PSY than in C. This suggests the enhanced activity in the HPA axis of PSY under baseline conditions. Additionally, enhanced Fos expression in PVN was found in PSY, compared with C, under baseline conditions. The elevated activity in the PVN neuron of PSY suggests that PSY will have a constitutively enhanced activity in the HPA system. At PFC, AMY, and LC, no significant group differences were detected. This enhanced activity in the HPA system in rats with prenatal psychological stress could be related to the elevated emotionality in the open field test in the present study. Based on the present study, we suggest that prenatal stress given psychologically with little physical stress could bring about changes in emotionality, depression-like behavior, and neurobiological mechanisms. We have assumed that the intensity of psychological stress in the present study would be very weak, compared with previous studies with physical stress. However, abnormality in the behavior and neurobiology in the offspring with prenatal psychological stress corresponded to the abnormality in rats with conventional physical stress. It was reported that the increases in brain noradrenaline release induced by chronic physical stress were rapidly reduced, while those caused by chronic psychological stress were enhanced (Tanaka, 1999). That is, the strength of chronic psychological stress might be comparable with or perhaps even stronger than that of chronic physical stress. This may explain the abnormality, which was greater than expected, in the offspring with prenatal psychological stress. Furthermore, it was reported that this psychological stress in the communication box could cause changes in the brain monoamine system, immune functions, and body temperature and that these changes were different from those brought about by physical stress (Endo and Shiraki, 2000; Noguchi et al., 2001; Oishi et al., 2003). Further studies should be done to clarify the differences in postnatal consequences between physical and psychological stress given prenatally. Acknowledgement We thank Miss Fumiko Tsuda for her technical assistance with this work. References Bermack, J.E., Debonnel, G., 2005. The role of sigma receptors in depression. J. Pharmacol. Sci. 97, 317–336. Bhatnagar, S., Lee, T.M., Vining, C., 2005. Prenatal stress differentially affects habituation of corticosterone responses to repeated stress in adult male and female rats. Horm. Behav. 47, 430–483. Chapillon, P., Patin, V., Roy, V., Vincent, A., Caston, J., 2002. Effects of pre- and postnatal stimulation on developmental, emotional, and cognitive aspects in rodents: a review. Dev. Psychobiol. 41, 373–387. Crusio, W.E., 2001. Genetic dissection of mouse exploratory behaviour. Behav. Brain Res. 125, 127–132.
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Prenatal psychological stress causes higher emotionality, depression-like behavior, and elevated activity in the hypothalamo-pituitary-adrenal axis Hiroshi Abe a,*, Noriko Hidaka a, Chika Kawagoe a, Kei Odagiri a, Yuko Watanabe a, Testuya Ikeda b, Yuta Ishizuka a, Hiroyuki Hashiguchi a, Ryuichiro Takeda a, Toshikazu Nishimori b, Yasushi Ishida a a
Division of Psychiatry, Department of Clinical Neuroscience, School of Medicine, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan b Division of Neurobiology, Department of Anatomy, School of Medicine, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan Received 21 February 2007; accepted 14 June 2007 Available online 19 June 2007
Abstract In humans, stressful events during pregnancy may raise the risk of psychiatric disorders in offspring, and studies with rodents have found that physical prenatal stress can cause changes in the physiology, neurobiology, and behavior of offspring. In the present study, we examined whether psychological prenatal stress with little physical stress could cause changes in the neurobiology and behavior of offspring in Sprague–Dawley rats, as physical prenatal stress did. Dams received psychological stress by observing a rat being electrically shocked behind a transparent wall in the social communication box during the last trimester of gestation but were not exposed to any physical stress. Male offspring from the dams exposed to psychological stress showed enhanced emotionality in an open field test, depression-like behavior in a forced swim test, and enhanced activity in the hypothalamo-pituitary-adrenal axis, compared with rats from untreated dams. However, the prenatally stressed rats showed intact ability to acquire context conditioning. This is the first report that psychological prenatal stress in the communication box can cause changes in the neurobiology and behavior of offspring in rodents. # 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Prenatal psychological stress; Anxiety; Depression; Fos immunohistochemistry; Hypothalamic-pituitary-adrenal axis; Communication box
1. Introduction In humans, it has been reported that stressful events during pregnancy may raise the risk of psychiatric disorders in offspring (Wadhwa et al., 2001; Weinstock, 2001; Maccari et al., 2003). Studies with rodents give us a clearer relationship between stressful treatment during pregnancy and changes in the physiology and behavior of the offspring (Kofman, 2002; Maccari et al., 2003; Weinstock, 2005). Generally, prenatally stressed rats showed higher emotional reactivity, higher levels of anxiety, and depression-like behavior. In such studies, physical stress was given to the dams. For example, restraint stress, to which dams were exposed for 45 min three times per day with bright light during the last trimester of gestation, * Corresponding author. Tel.: +81 985 85 2969; fax: +81 985 85 5475. E-mail address: [email protected] (H. Abe).
has been most widely used in recent studies with rodents (Bhatnagar et al., 2005; Viltart et al., 2006; Wang et al., 2006). Additionally, foot shock (Estanislau and Morato, 2005), injection of saline (White and Birkle, 2001), loud noise (Wakshlak and Weinstock, 1990), or a combination of these (Koenig et al., 2005) were used as physical stress. However, in humans, we may say that pregnant women are exposed to psychological stress more frequently than to physical stress. Although pregnant women may experience physical injury caused by accidents or infections, they are more often exposed to psychological stress, such as anxiety about their babies and their entirely new lifestyle, financial problems, workplace worries, and so on. Therefore, to examine the etiology of psychiatric disorders caused by prenatal stress in humans, one should use an animal model, in which the effects induced by psychological stress but not physical stress can be evaluated. However, as stated above, to our knowledge, physical stress was
0168-0102/$ – see front matter # 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2007.06.1465
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given to the dams in almost all studies with rodents. In fact, Chapillon et al. (2002) reviewed a series of their studies with emotional stress. In their experiment, where they exposed dams to a cat as a prenatal stress, exposure to a cat was acutely carried out at gestational days 10, 14, or 19. On the other hand, we intended to expose dams to psychological stress during the last trimester of gestation to compare directly the effect of conventional restrain stress procedure (Bhatnagar et al., 2005; Viltart et al., 2006; Wang et al., 2006). Furthermore, according to their description (Lordi et al., 1997), when dams were put in a box with a cat together, ‘‘rats exhibited strong motor and autonomic reactions’’. This suggested that dams and fetus would have considerable physical stress as well as psychological stress. Moreover, intensity of stress would be difficult to control because a cat put in a box would not behave against each pregnant rat in a similar manner. Therefore, in this study, we used psychological stress produced by the social communication box (Ogawa and Kuwabara, 1966). The communication box is an apparatus in which subjects observe a rat being electrically shocked behind a transparent wall. This seems to expose subjects to psychological stress with no physical stress. To examine emotional reactivity, depression-like behavior, and learning ability of male offspring from the dams exposed to psychological stress, comparing with offspring from untreated dams, rats were tested in an open field test, a forced swim test, and a context conditioning. Additionally, neurobiological differences were examined by measuring plasma corticosterone contents and the number of Fos immunopositive cells in some brain regions between them. 2. Materials and methods 2.1. Animals Six Sprague–Dawley pregnant rats (9-week-old) at the 13th gestational day and six Sprague–Dawley female rats (10-week-old) were obtained from Charles-River, Japan, and were individually housed on a 12 h light/dark cycle (light on: 08:00 h) with free access to food and water. The pregnant rats were nulliparous until this pregnancy. The pregnancy was confirmed by the existence of the plug in the vaginas. Three pregnant females were given psychological stress and the others were not. Six nonpregnant female rats were used as ‘‘presenters’’ that actually received the electrical foot shock. The ethical committee for animal experimentation at University of Miyazaki approved the experimental protocols used.
2.2. Prenatal stress procedure A communication box with a grid floor composed of 5 mm diameter stainless steel rods placed 13 mm apart was used. It had four compartments (15 cm 15 cm) divided by transparent plastic walls. An electric shock generator (MSG-001, Toyo Sangyo, Japan) was controlled by a program of our own making. Electric foot shocks (0.3, 0.4, or 0.5 mA) for 1 s were randomly presented once every 60 s. There were plastic plates on the grid floor in three compartments and pregnant rats put in these compartments never had electric shocks, while the ‘‘presenter’’ rat was put in the compartment without a plastic plate. Three sessions for stress exposure were carried out for 60 min at 09:00, 12:00, and 14:00 h in 1 day. These stress sessions were carried out during gestational day 13–20. The presenter rat was changed every session, so each presenter rat was used every six sessions. All six pregnant females were transported to a waiting room from home cages at an animal center every day and handled to weigh their body weight. Three pregnant rats were given psychological stress in above procedure after transferring from the waiting room to an experiment room. The other three dams were kept in the waiting
room, and they were not given psychological stress. The pups were weaned at 21 days of age and housed in groups of three or four in a cage. Only male rats were used in this study. Offspring from the dams exposed to psychological stress were used as the prenatally, psychologically stressed (PSY) group, and offspring from the dams without any treatment were used as a control (C) group. All rats performed open field test, but different animals were used in other behavioral tasks to suppress the confounding factors to the minimum, because discomfort experience in the context conditioning might affect the performances in the forced swim test carried out in the same laboratory.
2.3. Open field test All rats in groups C (n = 20) and PSY (n = 20) were tested in an open field arena at postnatal day (PND) 60. The open field apparatus was a square box (100 cm 100 cm 40 cm) constructed of gray Plexiglas. Testing was conducted under dim light (about 80 lx). The rat’s behavior was recorded using a video camera and analyzed from the videotapes, using the motion tracking system (Dipp-Motion 2D, DITECT, Japan). The rat was placed in the center of the arena and behaved freely for 5 min. The numbers and durations of the following behavioral parameters were recorded during 5 min: rearing, standing with the body inclined vertically, forequarters raised with and without its forelimb touching the wall of the apparatus; the total time spent in the center square (33 cm 33 cm); the total time spent in the four corners (12 cm 12 cm at each corner); traveling speed and total distance traveled; exploration, at-least-once entered open field units, that is, the area covered by rats during a 5 min period. Scores ranged from 0 to 9. The number of rearings was manually counted by experimenters, and other indexes were analyzed with the above motion tracking system.
2.4. Context conditioning Rats in groups C (n = 6) and PSY (n = 6) were tested in the context conditioning at PND 110. They were tested in the open filed test at PND 60, and they came from all six litters (two rats per litter) The testing apparatus was a shock box (29 cm 26 cm 28 cm) with a stainless-steel bar floor placed in an isolation chamber with a semitransparent window to allow behavioral observation. A mild foot shock (0.3 mA; 60 Hz) was generated with a shock generator using a scrambler (Toyo Sangyo Co., Toyama, Japan) and delivered through the grid chamber floor. The timing of the shocks was controlled with a repeated-cycle timer; they lasted 1 s and were delivered every 15 s for 20 min. Rats were re-exposed to the testing apparatus in which they had received the foot shock treatment 1 day before. The duration of immobile behavior during 10 min and the latency until the first movement were recorded. The duration of immobility, defined as the animal being motionless but alert, was measured by directly observing the motion of each animal during the 10 min period in the testing apparatus in the PSY and C groups.
2.5. Forced swim test A modified version of the forced swim test (Porsolt et al., 1977, 1978) was performed by rats in groups C (n = 6) and PSY (n = 6) at PND 120. They came from all six litters (two rats per litter) and tested in the open field test at PND 60, but did not experience the context conditioning at PND 110. A water tank (height = 35 cm; diameter = 30 cm) was filled with 20 8C water up to a level of 22 cm. In the first day, rats were placed in the water tank for 15 min. The water was changed for every rat. Rats were again placed in the water tank for 5 min on the next day. The duration of immobility behavior (floating in water without active movements of forepaws) was measured by experimenters.
2.6. Immunohistochemical analysis Rats in groups C (n = 6) and PSY (n = 5) which performed the open field test at PND 60 but did not experience context conditioning or the forced swim test were used. They were deeply anesthetized at PND 130 with sodium pentobarbital and perfused transcardially, first with heparinized saline, then with 500 ml of cold, freshly prepared 4% paraformaldehyde, and finally with 500 ml of 10% sucrose in 0.1 M phosphate buffer. The brains were removed immediately, immersed in 30% sucrose in 0.1 M phosphate buffer for 2 days at 4 8C,
H. Abe et al. / Neuroscience Research 59 (2007) 145–151 and then cut coronally into 50 mm sections on a freezing microtome for immunohistochemical examination. From each animal section through the prefrontal cortex (PFC; approximately 3.2 mm rostral to the bregma), the hypothalamic paraventricular nucleus (PVN; approximately 1.8 mm caudal to the bregma), the amygdala (AMY; approximately 2.8 mm caudal to the bregma), the dorsal raphe nucleus (DR; approximately 7.8 mm caudal to the bregma), and the locus coeruleus (LC; approximately 10.0 mm caudal to the bregma) were selected for histological study (Paxinos and Watson, 1998). The sections were collected in a phosphate-buffered saline solution (PBS; pH 7.4) and processed for Fos immunohistochemistry according to the manufacturer’s instructions for use with the streptavidin–biotin system (Histofine SAB-PO (R) kit, Nichirei, Tokyo, Japan). After incubation in 10% normal goat serum for 20 min, the sections were incubated at 4 8C overnight with a Fos antibody, a rabbit polyclonal antibody raised against a peptide corresponding to human c-fos amino acid residues 3–16 (diluted 1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). They were then rinsed three times in PBS and incubated at room temperature for 45 min with a secondary biotinylated goat antirabbit IgG, again rinsed three times in PBS, and further incubated at room temperature for 15 min with a streptavidin–peroxidase complex. After three rinses in PBS, the reaction products of biotinylated goat antirabbit IgG and streptavidin-conjugated horseradish peroxidase were intensified by pretreatment with 0.125% cobalt chloride, then visualized using 0.01% diaminobenzidine tetrahydrochloride and 0.0003% hydrogen peroxide. The sections were mounted on gelatin-coated glass slides, air-dried, dehydrated, cover-slipped, and Fos immunoreactivity was analyzed using light microscopy. The two most heavily labeled sections through each structure of each animal were chosen for cell counting. The number of Fos immunopositive cells was counted in two sections per structure in each animal in a standardized manner under 200 magnification using a microscopic 0.25 mm 0.25 mm grid. The grid was placed in the center of the eye piece. The average number of Fos immunopositive cells in each structure was computed.
2.7. Plasma corticosterone assay A blood sample (1 ml) was collected from the right atrium under deep anesthesia before the perfusion described above. Heparin (10 ml) was mixed into a sample of collected blood. This sample was centrifuged at 6100 g at 4 8C for 25 min, and the supernatant was diluted with a buffer supplied by the manufacturer. Plasma corticosterone content was measured by ELISA using a commercially available kit (rodent corticosterone ELISA test kit, Endocrine Technologies Inc., USA). ELISA was performed in a 96-well plate, where antibodies were coated. Standards and samples were pipetted into appropriate wells, and a solution of corticosterone labeled with horse radish peroxidase was added to each well. After incubation for 2 h at 37 8C, the reaction was terminated by washing the plate with wash solution. Colorimetric detection of peroxidase activity was achieved by adding tetramethylbenzidine solution and incubating for 20 min at room temperature. The enzymatic reaction was stopped with stopping solution, and the optical density of each well was measured at 450 nm using a plate reader (MTP-500, Corona Electric Co. Ltd, Japan). A standard curve was produced by using six standard values, in which the absorbency values for blank tubes were subtracted. Results for the samples were read from this standard curve by a computer program.
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exposed to stress and dams not exposed to stress were 305.8 15.6 and 309.1 14.8, respectively. On the last day for the stress exposure, they were 349.8 16.2 and 358.0 17.9, respectively. That is, gains of body weights of dams exposed to psychological stress were slightly lower than dams without stress exposure. However, the differences were not significant. Body weights of offspring were analyzed at PND 60 and 105. There were no significant difference in the body weights between PSY (278.3 3.7) and C (270.3 5.0) at PND 60 [F(1,38) = 1.67, p = 0.20]. At PND 105, the body weights of PSY (460.0 5.8) tended to be greater than C (437.4 10.1) [F(1,37) = 3.9, p < 0.1]. One rat of C group was found dead in the morning at PND 61, and cause of the death was uncertain. 3.2. Open field test There was no significant difference in the total time spent in the center area between PSY and C [F(1,38) = 1.14, p = 0.29] (Fig. 1A). On the other hand, rats in group PSY spent significantly more time in the corners of the arena than those in C [F(1,38) = 6.74, p < 0.05] (Fig. 1B). PSY showed significantly less distance traveled during the 5 min session than C [F(1,38) = 5.98, p < 0.05] (Fig. 1C). Furthermore, the moving speed was also lower in PSY than in C [F(1,38) = 4.46, p < 0.05] (Fig. 1D). The number of rearings in PSY was significantly less than in C [F(1,38) = 4.32, p < 0.05] (Fig. 1E). PSY explored a significantly lower number of divisions in the open field arena than C [F(1,38) = 10.06, p < 0.005] (Fig. 1F).
2.8. Statistical analysis One-way ANOVAs were used to assess the differences among measurements in the open field test, context conditioning, and forced swim test. The body weights, the number of Fos immunopositive cells in each brain area, and the level of plasma corticosterone in the C and PSY groups were analyzed by one-way ANOVA.
3. Results 3.1. Body weights of dams and offspring Body weights of dams during stress exposure were analyzed. On the initial day, mean body weights standard error of dams
Fig. 1. Effects of prenatal psychological stress on the open field test. (A) Time spent in the center square (33 cm 33 cm) and (B) in the corners (12 cm 12 cm for each corner). (C) Total distance traveled in the arena. (D) Moving speed. (E) Number of rearings. (F) Number of divisions explored. Data are expressed as means + S.E.M. **p < 0.005, *p < 0.05.
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H. Abe et al. / Neuroscience Research 59 (2007) 145–151 Table 1 Number of Fos immunopositive cells in five brain areas of C and PSY Area
PFC PVN AMY DR LC
Treatment C
PSY
4.9 2.8 17.9 4.1 10.3 3.5 9.2 3.6 21.9 6.0
18.3 8.9 34.3 3.6* 7.1 1.7 23.6 4.1* 38.6 9.6
Value represents the mean number S.E.M. PFC: prefrontal cortex, PVN: hypothalamic paraventricular nucleus, AMY: amygdala, DR: dorsal raphe nucleus, and LC: locus coeruleus. Significant differences between PSY and C were detected in PVN and DR (*p < 0.05).
LC: F(1,9) = 2.34, p = 0.16]. In PVN (Fig. 4) and DR, the number of Fos immunopositive cells was significantly higher in PSY than in C [PVN: F(1,9) = 8.56, p < 0.05; DR: F(1,9) = 7.18, p < 0.05]. Fig. 2. Effects of prenatal psychological stress on the context conditioning. (A) Latency of the first movement and (B) duration of immobility behavior in the shock box. Data are expressed as means + S.E.M. No significant difference was detected.
3.3. Context conditioning The duration of immobility during 10 min (Fig. 2A) and the latency of the initial movement (Fig. 2B) were analyzed. There were no significant differences between these measurements for PSY and C [F(1,10) = 0.99, p = 0.34, F(1,10) = 2.79, p = 0.13, respectively]. 3.4. Forced swim test In the forced swim test, the duration of immobility during the 5 min test session was significantly longer in PSY than in C [F(1,10) = 6.61, p < 0.05] (Fig. 3). 3.5. Immunohistochemistry (Table 1) In PFC, AMY, and LC, there was no significant difference in the number of Fos immunopositive cells between PSY and C [PFC: F(1,9) = 2.41, p = 0.15; AMY: F(1,9) = 0.61, p = 0.45;
Fig. 3. Effects of prenatal psychological stress in the forced swim test. Duration of immobility behavior in test session. Data are expressed as means + S.E.M. *p < 0.05.
Fig. 4. High-magnification, bright-field photomicrographs showing the enhancement of Fos immunopositive cells in the paraventricular hypothalamic nucleus (PVN) of C and PSY groups. The square indicates the region in which the number of Fos immunopositive cells was counted. 3V: third ventricle. Scale bar = 100 mm.
H. Abe et al. / Neuroscience Research 59 (2007) 145–151 Table 2 Basal plasma corticosterone in PSY and C Treatment
Plasma corticosterone content
C PSY
89.3 24.6 172.0 18.1*
Value represents the mean concentration S.E.M. (ng/ml). The basal level of plasma corticosterone of PSY was significantly higher than that of C (*p < 0.05).
3.6. Plasma corticosterone assay (Table 2) The basal level of plasma corticosterone was significantly higher in PSY than in C [F(1,9) = 6.70, p < 0.05]. 4. Discussion In the present study, we demonstrated that psychological stress given to pregnant dams could cause enhanced emotionality, depression-like behavior, and enhanced activity in the hypothalamo–pituitary–adrenal (HPA) system of offspring in rats. In this study, the dams delivering C groups were not placed in the novel communication box, while the dams delivering PSY groups were placed in such a box. Therefore, exposure to the novel environment, in addition to the exposure to shocked rats, may produce the effects between two groups. However, in any case, exposed stress mainly involved much psychological factor and less physical factor, comparing restraint stress (Bhatnagar et al., 2005; Viltart et al., 2006; Wang et al., 2006), which is most widely used as prenatal stress procedure. The gains of body weights of dams exposed to psychological stress were slightly lower than dams without stress exposure. However, the differences were not significant. Some researches have reported that prenatal stress reduce body weight of offspring (Herrenkohl, 1979; Rhees and Fleming, 1981; Pollard, 1984), however such reduction of body weight was not found at PND 60 and 105. Furthermore, any prenatally stressed rats did not die through this experiment. In these respects, the effect of psychological stress exposed to the dams in this experiment would be milder than the stress provided by conventional procedures. In an open field test, rats are exposed to contradictory motivations to avoid aversive stimuli (novelty, lit, and open area) and to explore new places (Crusio, 2001). The internal conflict is assumed from some behavioral measurements. For example, the wall-seeking behavior, which is measured by duration to stay the peripheral area near the wall and to avoid the center area in the present study, is called thigmotaxis, and more thigmotactic behavior is considered to reflect more emotionality (anxiety or fear) because this behavior may reflect phylogenetically prepared fear reactions to avoid predators (Treit and Fundytus, 1989). This thigmotaxis has been used to measure fear or anxiety in open field tests. On the other hand, the distance traveled, the number of rearings, and the number of at-least-once-entered open field units has been thought to reflect animals’ exploratory tendencies. In the present study, there was no significant difference in the duration spent in the center
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area between the C and PSY groups, while PSY showed significantly more time spent in the corners, less distance traveled, fewer rearings, and fewer at-least-once-entered open field units than C. These results suggest that PSY should show higher emotionality and lower tendency to explore than C. Elevated emotionality has been reported in findings using prenatally, physically stressed rats in an open field test (Wakshlak and Weinstock, 1990; Poltyrev et al., 1996; Van den Hove et al., 2005). Furthermore, enhanced emotionality of prenatally stressed rats has been also reported in the elevated plus maze task (Wakshlak and Weinstock, 1990), a task often used to examine an animal’s fear or anxiety (Pellow and File, 1986). The present result suggests that even psychological stress with little physical stress to pregnant dams could change emotionality in offspring. In the context conditioning, the ability of rats to learn and remember an association between an aversive stimulus and contextual cues can be examined (Fanselow, 1980). There were no significant differences in the latency of initial movement and the duration of freezing between the C and PSY groups. Some researchers reported that severe prenatal physical stress disrupted this conditioning (Fride et al., 1986; Shiota and Kayamura, 1989), while mild stress enhanced the conditioning (Fujioka et al., 2001). On the other hand, the present results suggest that rats whose mother was exposed to psychological stress will not have deficits in learning and memory about an association between an aversive stimuli and context. Additionally, the number of Fos immunopositive cells in the amygdala, which is considered to be closely involved with context conditioning (Fanselow and Gale, 2003), did not differ significantly between PSY and C. Thus, the prenatal psychological stress in this study may not have been strong enough to cause any behavioral and neurobiological changes in the conditioning. In the forced swim test, the time spent swimming and the time spent floating in a cylinder filled with water were measured (Porsolt et al., 1977, 1978). The present results indicate that rats become more depressed because immobility increased and struggling behavior decreased. The validity of this test is recognized to some extent because the immobility behavior is decreased by many types of antidepressants and increased by stressors including immobilization, foot shock, and intruder encounters (Weiss et al., 1981; Zebrowska-Lupina et al., 1990; Hebert et al., 1998). Some studies following physical prenatal stress (Morley-Fletcher et al., 2003, 2004) demonstrated increased immobility behavior, suggesting that prenatally stressed rats will be more depressed than normal rats. The results of the present study suggest that prenatal stress given psychologically with little physical stress could make rats more depressed than untreated rats. Serotonergic neurons in the dorsal raphe nucleus are considered to play important roles in depression (Bermack and Debonnel, 2005). In the present study, the number of Fos immunopositive cells in DR of PSY was significantly higher than that in C. This suggests that depression-like behavior shown in PSY was related to the enhanced activity of serotonergic neurons of DR. Prenatally stressed rats, compared with normal rats, usually showed elevated level of plasma corticosterone (Weinstock
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et al., 1998; Maccari et al., 2003). For example, Weinstock et al. (1998) found a significantly elevated level of corticosterone at the baseline (before foot shock) and 15 min after foot shock in rats with prenatal stress. Correspondingly, in the present study, the basal level of plasma corticosterone was significantly higher in PSY than in C. This suggests the enhanced activity in the HPA axis of PSY under baseline conditions. Additionally, enhanced Fos expression in PVN was found in PSY, compared with C, under baseline conditions. The elevated activity in the PVN neuron of PSY suggests that PSY will have a constitutively enhanced activity in the HPA system. At PFC, AMY, and LC, no significant group differences were detected. This enhanced activity in the HPA system in rats with prenatal psychological stress could be related to the elevated emotionality in the open field test in the present study. Based on the present study, we suggest that prenatal stress given psychologically with little physical stress could bring about changes in emotionality, depression-like behavior, and neurobiological mechanisms. We have assumed that the intensity of psychological stress in the present study would be very weak, compared with previous studies with physical stress. However, abnormality in the behavior and neurobiology in the offspring with prenatal psychological stress corresponded to the abnormality in rats with conventional physical stress. It was reported that the increases in brain noradrenaline release induced by chronic physical stress were rapidly reduced, while those caused by chronic psychological stress were enhanced (Tanaka, 1999). That is, the strength of chronic psychological stress might be comparable with or perhaps even stronger than that of chronic physical stress. This may explain the abnormality, which was greater than expected, in the offspring with prenatal psychological stress. Furthermore, it was reported that this psychological stress in the communication box could cause changes in the brain monoamine system, immune functions, and body temperature and that these changes were different from those brought about by physical stress (Endo and Shiraki, 2000; Noguchi et al., 2001; Oishi et al., 2003). Further studies should be done to clarify the differences in postnatal consequences between physical and psychological stress given prenatally. Acknowledgement We thank Miss Fumiko Tsuda for her technical assistance with this work. References Bermack, J.E., Debonnel, G., 2005. The role of sigma receptors in depression. J. Pharmacol. Sci. 97, 317–336. Bhatnagar, S., Lee, T.M., Vining, C., 2005. Prenatal stress differentially affects habituation of corticosterone responses to repeated stress in adult male and female rats. Horm. Behav. 47, 430–483. Chapillon, P., Patin, V., Roy, V., Vincent, A., Caston, J., 2002. Effects of pre- and postnatal stimulation on developmental, emotional, and cognitive aspects in rodents: a review. Dev. Psychobiol. 41, 373–387. Crusio, W.E., 2001. Genetic dissection of mouse exploratory behaviour. Behav. Brain Res. 125, 127–132.
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