- Email: [email protected]
Prenatal stress induces anxiety-like behavior together with the disruption of central serotonin neurons in mice
Neuroscience Research 70 (2011) 111–117
Contents lists available at ScienceDirect
Neuroscience Research journal homepage: www.elsevier.com/locate/neures
Rapid communication
Prenatal stress induces anxiety-like behavior together with the disruption of central serotonin neurons in mice Kazuya Miyagawa, Minoru Tsuji ∗ , Kanji Fujimori, Yasuho Saito, Hiroshi Takeda ∗∗ Division of Pharmacology, Department of Pharmaceutical Sciences, School of Pharmacy, International University of Health and Welfare, 2600-1 Kitakanamaru, Ohtawara, Tochigi 324-8501, Japan
a r t i c l e
i n f o
Article history: Received 28 October 2010 Received in revised form 19 January 2011 Accepted 7 February 2011 Available online 12 February 2011 Keywords: Serotonin Prenatal stress Neurodevelopment Anxiety Emotional behavior
a b s t r a c t Most pregnant women are at risk of showing some emotional abnormality, since some biological functions such as hormonal systems may dramatically change in pregnancy. Some of them may be exposed to strong stress as hesitation of positive drug therapies because of worries regarding adverse effects on the embryo. A growing body of evidence suggests that prenatal stress increases the vulnerability to neuropsychiatric disorders, including depression and anxiety. However, the mechanisms involved are still unknown. To clarify the influence of exposure to prenatal stress on emotional development, we examined behavioral responses in offspring exposed to weak- or strong-prenatal restraint stress. We found that offspring that had been exposed to strong stress displayed anxiety-like behavior as determined by the elevated plus-maze test. It has been widely accepted that central serotonin (5-hydroxytryptamine; 5-HT) neurons play a critical role in emotional behaviors. Immunohistochemical studies showed that exposure to strong-prenatal restraint stress increased the expression of 5-HT-positive cells in the dorsal raphe nuclei in mice. Moreover, under these conditions, tryptophan hydroxylase-like immunoreactivities were also dramatically increased. In contrast, these behavioral and neurochemical abnormalities were not observed in offspring that had been exposed to weak-prenatal restraint stress. These findings indicate that exposure to excessive prenatal stress induces anxiety-like behavior together with disruption of the development of 5-HT neurons in mice. © 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Depression and anxiety disorders are common public health problems with a lifetime prevalence of 10–20%, yet the mechanisms that underlie their pathophysiology are still poorly understood. Most pregnant women are at risk of showing some emotional abnormality, since some biological functions such as hormonal systems may dramatically change in pregnancy. Indeed, it has been reported that 16% of women experienced the onset of an affective disorder during pregnancy, and 68% of them showed symptoms during the first trimester (Kitamura et al., 1993). Thus, a suitable management plan that includes drug therapy may be necessary for pregnant women. However, doctors hesitate to use positive drug therapies because of worries regarding adverse effects on the embryo. Inappropriate mental support for pregnant women may result in their exposure to very stressful situations. Particularly when pregnant women suffer from serious mental disease, inappropriate drug therapies would expose them to the very severe
∗ Corresponding author. Tel.: +81 287 24 3489; fax: +81 287 24 3521. ∗∗ Corresponding author. Tel.: +81 287 24 3436; fax: +81 287 24 3521. E-mail addresses: [email protected] (M. Tsuji), [email protected] (H. Takeda).
stressful situations. Previous clinical research on pregnant women suffering depressive disorders indicated that the activity of the fetus and newborn is elevated, prenatal growth is delayed, and prematurity and low birthweight are more frequent (Field et al., 2006). Moreover, it has also been suggested that exposure to stress during gestation may impair the emotional development of the offspring and as a result the incidence of numerous neuropsychiatric disorders, including depression, anxiety, schizophrenia, and autism, may increase (Gillott and Standen, 2007; Koenig et al., 2002; Walker et al., 2008). Additionally, recent preclinical studies have suggested that prenatal stress induces several functional and structural abnormalities of the components that regulate stress responses, such as the hypothalamic–pituitary–adrenal (HPA)-axis (Lephart et al., 1997; Szuran et al., 2000) or monoamine neurotransmission (Peters, 1982, 1990; Weinstock, 1997). Moreover, the offspring exposed to prenatal stress show the abnormal psychiatric behaviors such as the increased fear and anxiety (Nishio et al., 2001; Vallée et al., 1997), persistent paradoxical sleep alterations (Dugovic et al., 1999), deficits of learning and memory (Hayashi et al., 1998; Vallée et al., 1999), depressive-like behavior (Secoli and Teixeira, 1998; Zimmerberg and Blaskey, 1998; Alonso et al., 1999) and schizophrenia-like behavior (Lehmann et al., 2000). However,
0168-0102/$ – see front matter © 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2011.02.002
112
K. Miyagawa et al. / Neuroscience Research 70 (2011) 111–117
the mechanisms accounting for the impact of the prenatal stress on the life of adulthood have not fully understood. On the other hand, axons from the neurons of the raphe nuclei, the principal source of serotonin (5-hydroxytryptamine; 5-HT) release in the brain, form a neurotransmitter system, reaching almost every part of the central nervous system. Recent clinical and preclinical studies have suggested that central 5-HT neurotransmission may be involved in the aetiology, expression and treatment of anxiety, impulsiveness and depression (Murphy and Pigott, 1990). In addition, it has been widely accepted that several medicines for psychiatric disorders were designed as mediator to 5-HT neurotransmission. 5-HT furthermore acts as a growth factor during embryogenesis, and serotonin receptor activity forms a crucial loop in the pathway leading to changes in brain structure (Sodhi and Sanders-Bush, 2004). Therefore, the incidence of numerous neuropsychiatric disorders caused by the exposure to stress during gestation might be associated with the disruption of 5-HT neurotransmission. Actually a few preclinical reports suggest that prenatal stress could affect on the central 5-HT neuron (Peters, 1982, 1990; Hayashi et al., 1998). On the basis of the previous reports, the aim of the present study was to clarify the influence of exposure to prenatal stress on the development of emotionality and central 5-HT neurons. The present studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the Committee on the Care and Use of Laboratory Animals of the International University Health and Welfare. All experiments were performed using 7–10-week-old male offspring of ICR mice (Japan SLC, Inc., Shizuoka, Japan) that had been prenatally exposed to stress as described below, or naive mice. The mice were housed at a room temperature of 23 ± 1 ◦ C and humidity of 50 ± 5% with a 12 h light–dark cycle (light on 7:00 a.m.–7:00 p.m.). Food and water were available ad libitum. Virgin female mice were mated at 10–11 weeks of age. The presence of a copulation plug denoted gestation day (GD) 0.5. Pregnant females were housed individually. In the present study, we used two types of prenatal stress model modified on the basis of previous reports (Chung et al., 2005; Van den Hove et al., 2005). In the model mice exposed to weak-prenatal restraint stress (WPRS), the pregnant mice were placed in a 50 (for mice with a body weight of 40 g or less)- or 70 (for mice with a body weight of 40 g or more)ml polystyrene tube three times a day (at approximately 10:00 a.m., 14:00 p.m., and 18:00 p.m.) for 1 h from GD 5.5 to GD 17.5. On the other hand, in the model mice exposed to strong-prenatal restraint stress (SPRS), the pregnant mice were placed in a tube as mentioned above at 6 h (10:00 a.m.–4:00 p.m.) a day. To investigate the changes in anxiety-like behaviors, 7–8 weeks old male offspring were tested using the elevated plus-maze paradigm (Muromachi Kikai Co., Ltd., Tokyo, Japan). The apparatus was elevated 40 cm from the ground and the maze consisted of two opposing open arms (30 cm × 6 cm × 0.3 cm) and two opposing enclosed arms (30 cm × 6 cm × 15 cm) that were connected by a central platform (6 cm × 6 cm, 40 lux), thus forming the shape of a plus sign. Each animal was placed on a central platform, and the distance that the mouse moved in the maze was recorded for 5 min by an overhead color CCD camera that tracked the centre of the mouse. Moreover, the time spent in and the numbers of entry into open or enclosed arms were also recorded. Data from the CCD camera were collected through a custom-designed interface (CAT-10; Muromachi Kikai) as a reflection signal. All of the data were analyzed and stored in a personal computer using analytical software (Comp ACT HBS, Muromachi Kikai). The results were calculated as mean ratios of the time spent in the open arms to the total time spent in both the open and enclosed arms. Entries into the open arms (%), total entries, moving distance (cm) and moving speed (cm/s) were also scored.
After elevated plus-maze paradigm, the mice were immediately placed in the center of open field (Muromachi Kikai) and the behavior of the mice in the open field was recorded for 5 min. The apparatus of open field paradigm was made of gray wooden box (50 cm × 50 cm × 50 cm, 170 lux). An infrared beam sensor was installed on the wall to detect the numbers and duration of rearing behaviors. Other behavioral performance, such as locus and the distance of movement (total locomotor activity (cm)) of mice, was recorded by an overhead color CCD camera. The head of the mice were painted yellow and the color CCD camera followed the center of gravity. Data from the CCD camera were collected through a custom-designed interface (CAT-10; Muromachi Kikai) as a reflection signal. All of the data were analyzed and stored in a personal computer using analytical software (Comp ACT HBS, Muromachi Kikai). The results were calculated as thigmotaxis (amount of time in the outer versus inner zone (33 cm × 33 cm)). The other parameters such as rearing behavior (count and duration), moving distance and moving speed (cm/s) were also scored. In the immunohistochemical analysis, mice were deeply anesthetized with sodium pentobarbital (70 mg/kg, i.p.) and perfusion-fixed with 4% paraformaldehyde (Wako Pure Chemical Industries Ltd., Osaka, Japan) in PBS. The brains were post-fixed in 4% paraformaldehyde in PBS for 12 h at 4 ◦ C. Brain sections (80 m thick) were prepared on a Microslicer (DTK-1000; Ted Pella, Inc., CA). The sections were incubated with 10% normal goat serum in ice-cold PBS for 60 min to block nonspecific antibody binding and then with the 5-HT rabbit monoclonal antibody (1:1000 dilution; Sigma Chemical, St. Louis, MO) or tryptophan hydroxylase (TPH) mouse monoclonal antibody (1:600; Sigma Chemical) for 2 days at 4 ◦ C. The samples were then rinsed with PBS and incubated with the appropriate secondary antibody conjugated with Alexa 488 (1:1000) for 12 h at 4 ◦ C. The sections were then rinsed with PBS and mounted on glass slides with 20% glycerol in PBS. Fluorescence immunolabeling was detected using a confocal laserscanning microscope (FV1000; Olympus Optical, Tokyo, Japan). The intensity of TPH-like immunoreactivity in dorsal raphe nuclei (DRN) was analyzed and quantified by computer-assisted densitometry using Image J software (National Institutes of Health, MD). The data are expressed as the mean with SEM. Statistical analyses were performed using Student’s t-test. The influence of prenatal exposure to stress on anxiety-like behaviors in mice was evaluated by the elevated plus-maze test. The results in mice that had been exposed to prenatal restraint stress are shown in Fig. 1. As shown in Fig. 1A and B, mice that had been exposed to WPRS show hypo-locomotion in the elevated plus-maze (Fig. 1A (moving distance); **p < 0.01 vs. control, Fig. 1B (number of total entries); **p < 0.01 vs. control). Under these conditions, WPRS-exposed mice failed to show the change in the percentage of entries into open arms (Fig. 1C) as well as percentage of time spent in open arms (Fig. 1D). Like WPRS-exposed mice, mice that had been exposed to SPRS show hypo-locomotion in the elevated plus-maze (Fig. 1F (moving distance); *p < 0.05 vs. control, Fig. 1G (number of total entries); ***p < 0.001 vs. control). On the other hand, the percentage of entries into open arms (Fig. 1H; **p < 0.01 vs. control) and the percentage of time spent in open arms (Fig. 1I; *p < 0.01 vs. control) were significantly decreased in SPRS-exposed mice. As another measurement of emotionality, the mice that had been prenatally exposed to restraint stress were evaluated by the open field test (Fig. 2). As shown in Fig. 2A–D, WPRS-exposed mice failed to show change in the thigmotaxis and general activity levels (total distance traveled and rearing) in open field. On the other hand, although SPRS-exposed mice failed to show change in the thigmotaxis (Fig. 2F), rearing count (Fig. 2G; **p < 0.01 vs. control), rearing duration (Fig. 2H; ***p < 0.001 vs. control) and moving distance (Fig. 2I; *p < 0.05 vs. control) were significantly
K. Miyagawa et al. / Neuroscience Research 70 (2011) 111–117
113
Fig. 1. Anxiety-like behaviors of mice that had been prenatally exposed to restraint stress as detected by the elevated plus-maze test. (A–E) Analysis in the WPRS-exposed mice. (F–J) Analysis in the SPRS-exposed mice. Each mouse placed on a central platform, and the distance that the mouse moved in the maze was recorded for 5 min. Moreover, the moving distance (A, F), the numbers of total entries into open and enclosed arms (B, G), the percentage of entries into open arms (C, H), percentage of time spent in the open arms (D, I) and moving speed (E, J) were scored. The representative images showing the mouse traveling patterns are shown in panel I. Each column represents the mean with SEM of 13-20 mice. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. Cont: control, WPRS: weak prenatal restraint stress, SPRS: strong prenatal restraint stress.
114
K. Miyagawa et al. / Neuroscience Research 70 (2011) 111–117
Fig. 2. General behaviors of mice that had been prenatally exposed to restraint stress as detected by the open field procedure. (A–E) Analysis in the WPRS-exposed mice. (F–J) Analysis in the SPRS-exposed mice. Each mouse was placed in the center of open field and the behavior of the mouse in the open field was recorded for 5 min. Moreover, the percentage of time spent in central area (A, F), the rearing counts (B, G) and duration (C, H), the moving distance (D, I) and moving speed (E, J) were scored. The representative images showing the mouse traveling patterns are shown in panel I. Rearing behaviors are represented as closed diamonds. Each column represents the mean with SEM of 13-20 mice. *p < 0.05, ***p < 0.001 vs. control. Cont: control, WPRS: weak prenatal restraint stress, SPRS: strong prenatal restraint stress.
K. Miyagawa et al. / Neuroscience Research 70 (2011) 111–117
decreased. These results indicate that exposure to excessive prenatal stress could induce the emotional abnormality, i.e., increase in anxiety levels in offspring. These results of behavioral studies suggest that SPRS-exposed mice could reflect the clinical case that the severe stressful situations during pregnancy impair the emotional development of offspring. The hypo-locomotion in the behavioral tests could be also observed if the mice exposed to prenatal stress presented motor dysfunction including delayed growth. In the present study, although we have not checked the motor functions using rota-rod test and so on, WPRS- and/or SPRS-exposed mice failed to show a clear motor deficit in their home cage and behavioral experiment apparatuses. Actually, the moving speeds were not significantly different in both experiment and in both group (Fig. 1Eand J, Fig. 2Eand J). Therefore, the hypo-locomotion observed in the present behavioral tests could indicate the emotional dysfunction induced by the exposure to prenatal restraint stress. The reason of why the hypo-locomotion of WPRS group was only observed by elevated plus-maze test could not be clearly explained. In our opinion, the fear level in the elevated plus maze paradigm is higher than that in the open field paradigm. Therefore, the present data suggest that although WPRS-exposed mice could show the abnormal emotional behavior in only powerful fear condition, SPRS-exposed mice could also show in the mild fear condition. Additionally, the reason of why that SPRS-exposed mice failed to show decrease in percent of time spent in central area in the open-field test also could not be clearly explained. One possible explanation is that the decrease in time spent in open area is observed only under the extreme stress condition, such as acute stress stimuli. Growing body of evidence suggests that brain 5-HT is implicated in the etiology of neuropsychiatric disorders, such as anxiety and depression (Murphy and Pigott, 1990). Most antidepressants or anxiolytics change the levels of 5-HT in the brain (Krishnan and Nestler, 2008). This suggests that biochemical imbalances within the central 5-HT systems may underlie the pathogenesis of these disorders. The DRN, a major source of 5-HT innervation of the forebrain, plays a critical role in stress responsiveness and the effects of antidepressant drugs commonly used to treat anxiety disorders (Sandford et al., 2000). Therefore, we next investigated the changes in 5-HT neurons in DRN of mice that had been prenatally exposed to restraint stress (Fig. 3). An immunohistochemical study showed that exposed to WPRS failed to change in the expression of 5-HTpositive cells (Fig. 3B–D). On the other hand, SPRS dramatically increased the expression of 5-HT-positive cells in DRN compared to the control (Fig. 3E–G; *p < 0.05 vs. control). To our knowledge, this is the first evidence that prenatal stress could induce structural abnormalities in the development of 5-HT neurons in DRN. We then investigated whether 5-HT neurons were functionally overexpressed in DRN of mice that had been prenatally exposed to restraint stress. Similar to 5-HT-positive cells, TPH-like immunoreactivities in DRN were also dramatically increased in SPRS-exposed mice compared with the control (Fig. 3K–M; *p < 0.05 vs. control), although such a neurochemical changes were not observed in WPRS-exposed mice (Fig. 3H–J). It has been widely accepted that TPH is the first-step and rate-limiting enzyme involved in the synthesis of 5-HT in the brain (Walther and Bader, 2003). Taken together, these results indicate that excessive prenatal restraint stress induces the overexpression of functional 5-HT neurons in DRN in mice. As mentioned above, a few preclinical reports suggest that prenatal stress could affect on the central 5-HT neurons. Peters (1990) reported that maternal stress increased fetal brain 5-HT synthesis in rat. They also found that the offspring exposed to prenatal stress showed region-specific changes in brain 5-HT, 5hydroxyindoleacetic acid (5-HIAA) and norepinephrine levels in infancy (Peters, 1982). Hayashi et al. (1998) reported that mater-
115
nal stress induced synaptic loss associated with disruption of 5-HT neurotransmission and developmental disabilities of offspring. In addition to these precedent articles, we here report a novel finding that the 5-HT positive neurons significantly increase in DRN of prenatal-stress mice. Although it has been widely accepted that brain 5-HT neuron play an important role in emotional behavior, its clear mechanism is not understood. There are various theories linking the function of 5-HT and its receptors to the actions of both anxiolytic and antidepressant drugs. As known in serotonin hypothesis, pharmacological manipulation to enhance 5-HT concentration in the brain increases anxiety (Handley and McBlane, 1993), in contrast, a reduction in 5-HT concentrations is associated with reduced anxiety. This suggests that anxiety is caused by abnormal increase in brain 5-HT concentrations. The present findings therefore suggest that prenatal stress may induces the superabundant brain 5-HT levels by overdevelopment of 5-HT neuronal cells, as a result, anxiety-like behavior in enhanced. Further studies should be needed to prove this hypothesis. Although the present findings do not fully explain the underlying mechanisms, they are supported by some recent preclinical studies. One of the potential mechanism by which stress can influence fetal development is the disturbance of hormonal systems in developmental stage. Especially, plasma estrogen and progesterone large increase during pregnancy (Tamby Raja et al., 1974). Moreover, prenatal stress could disrupt these hormonal systems (Field et al., 2006). To our knowledge the direct evidence that prenatal stress could affect 5-HT neurodevelopment via the disruption of these hormonal systems has not been reported. On the other hand, it has been recently indicated that these sex hormones interact with the 5-HT neuronal systems (Rivera et al., 2009; Uphouse et al., 2009). Additionally, the gender difference in 5-HT neurotransmission has also been reported (Kakeyama et al., 2002; Jovanovic et al., 2008). It is possible that sex hormones might be involved in mechanisms of present findings. The other potential mechanism is epigenetics, including DNA methylation (Seckl and Meaney, 2004; Tsankova et al., 2007; Weaver et al., 2004). Furthermore, attention has recently been focused on the involvement of epigenetic machinery in the programming of early life events. corticotropin releasing factor (CRF) and the glucocorticoid receptor (GR), which are important components involved in the stress response of the body, have both been previously shown to be regulated by methylation and are programmed early in life (Tsankova et al., 2007; Weaver et al., 2004). CRF is a known regulator of 5-HT neurotransmission (Bale and Vale, 2004; McGill et al., 2006; Tan et al., 2004; Valentino et al., 1991). During pregnancy, both plasma CRF and corticosteroid increase (Kammerer et al., 2006). Therefore, the dysregulation of CRF could be an upstream contributor to abnormalities in 5-HT transmission and a stress-sensitive phenotype. In fact, Mueller and Bale (2008) recently reported that changes in CRF and GR gene methylation correlated with altered gene expression, and thus provided important evidence of abnormal epigenetic programming during early prenatal stress. Therefore, it is expected that such epigenetic factors might play a role in the present findings. Furthermore, we should focus on the changes in functional molecules involved in the development of 5-HT neurons. 5-HT is an important neurotransmitter that has multiple functions in the central nervous system. Its synthesis is restricted to a very limited number of cells in the brainstem raphe nuclei with a vast axonal network. Alenia et al. (2006) reported that these cells express several markers of the serotonin lineage such as the rate-limiting enzyme in serotonin synthesis TPH2, the serotonin transporter, and the transcription factor Pet1. Pet1, LIM homeobox transcription factor 1b (Lmx1b), NK transcription factor related, locus 2 (Nkx2.2), Mouse achaete-scute homolog 1
116
K. Miyagawa et al. / Neuroscience Research 70 (2011) 111–117
Fig. 3. Morphological changes in 5-HT neurons in DRN of male mice that had been prenatally exposed to restraint stress. (A) Schematic diagram of the range of DRN enclosed by the line. (B–G) Typical photographs of 5-HT-positive cells in the DRN of control (B, E), WPRS-exposed (C) and SPRS-exposed (F) mice. The results of a quantitative analysis of 5-HT-positive cells in the DRN are shown in panels D and G. (H–M) Typical photographs of TPH-like immunoreactivity (IR) in the DRN of control (H, K), WPRS-exposed (I) and SPRS-exposed (L) mice. The results of a quantitative analysis of TPH-IR in the DRN are shown in panel J and M. The column represents the mean with SEM of 4–6 independent samples. *p < 0.05 vs. control mice. Scale bars: 200 m. Cont: control, WPRS: weak prenatal restraint stress, SPRS: strong prenatal restraint stress.
K. Miyagawa et al. / Neuroscience Research 70 (2011) 111–117
(Mash1), GATA-motif binding (Gata) 2, Gata3, and Paired-like homeodomain protein 2b (Phox2b) form a transcriptional network, which specifies the differentiation of serotonergic neurons around embryonic day 11 in the mouse. Recently, we found that prenatal stress also induces up-regulation of 5-HT-positive cells in the embryonic brain of mice (unpublished observation). Therefore, it is expected that stress stimuli could disrupt the transcriptional networks of 5-HT neurons across gestation. Further studies will be necessary to clarify the effects of prenatal stress on the development of 5-HT neurons in the embryonic brain. In summary, we found that excessive prenatal stress induced long-term emotional abnormalities associated with morphological and/or functional changes in central 5-HT neurons in mice. Furthermore, the disruption of 5-HT neuron development seems to also be induced by exposure to stress across gestation. The present findings provide evidence that prenatal stress is an important risk factor that underlies the induction of neuropsychiatric disorders in adulthood. References Alenia, N., Bashammakh, S., Bader, M., 2006. Specification and differentiation of serotonergic neurons. Stem Cell Rev. 2, 5–10. Alonso, S.J., Castellano, M.A., Quintero, M., Navarro, E., 1999. Action of antidepressant drugs on maternal stress-induced hypoactivity in female rats. Methods Find. Exp. Clin. Pharmacol. 21, 291–295. Bale, T.L., Vale, W.W., 2004. CRF and CRF receptors: role in stress responsivity and other behaviors. Annu. Rev. Pharmacol. Toxicol. 44, 525–557. Chung, S., Son, G.H., Park, S.H., Park, E., Lee, K.H., Geum, D., Kim, K., 2005. Differential adaptive responses to chronic stress of maternally stressed male mice offspring. Endocrinology 146, 3202–3210. Dugovic, C., Maccari, S., Weibel, L., Turek, F.W., Van Reeth, O., 1999. High corticosterone levels in prenatally stressed rats predict persistent paradoxical sleep alterations. J. Neurosci. 19, 8656–8664. Field, T., Diego, M., Hernandez-Reif, M., 2006. Prenatal depression effects on the fetus and newborn: a review. Infant. Behav. Dev. 29, 445–455. Gillott, A., Standen, P.J., 2007. Levels of anxiety and sources of stress in adults with autism. J. Intellect. Disabil. 11, 359–370. Handley, S.L., McBlane, 1993. 5HT drugs in animal models of anxiety. Psychopharmacology 112, 13–20. Hayashi, A., Nagaoka, M., Yamada, K., Ichitani, Y., Miake, Y., Okado, N., 1998. Maternal stress induces synaptic loss and developmental disabilities of offspring. Int. J. Dev. Neurosci. 16, 209–216. Jovanovic, H., Lundberg, J., Karlsson, P., Cerin, A., Saijo, T., Varrone, A., Halldin, C., Nordström, A.L., 2008. Sex differences in the serotonin 1A receptor and serotonin transporter binding in the human brain measured by PET. Neuroimage 39, 1408–1419. Kakeyama, M., Umino, A., Nishikawa, T., Yamanouchi, K., 2002. Decrease of serotonin metabolite in the forebrain facilitation of lordosis by dorsal raphe nucleus lesions in male rats. Endocr. J. 49, 573–579. Kammerer, M., Taylor, A., Glover, V., The HPA, 2006. axis and perinatal depression: a hypothesis. Arch. Womens Ment. Health. 9, 187–196. Kitamura, T., Shima, S., Sugawara, M., Toda, M.A., 1993. Psychological and social correlates of the onset of affective disorders among pregnant women. Psychol. Med. 23, 967–975. Koenig, J.I., Kirkpatrick, B., Lee, P., 2002. Glucocorticoid hormones and early brain development in schizophrenia. Neuropsychopharmacology 24, 309–318. Krishnan, V., Nestler, E.J., 2008. The molecular neurobiology of depression. Nature 455, 894–902. Lehmann, J., Stöhr, T., Feldon, J., 2000. Long-term effects of prenatal stress experiences and postnatal maternal separation on emotionality and attentional processes. Behav. Brain. Res. 107, 133–144. Lephart, E.D., Watson, M.A., Jacobson, N.A., Rhees, R.W., Ladle, D.R., 1997. CalbindinD28k is regulated by adrenal steroids in hypothalamic tissue during prenatal development. Brain. Res. Dev. Brain. Res. 100, 117–120.
117
McGill, B.E., Bundle, S.F., Yaylaoglu, M.B., Carson, J.P., Thaller, C., Zoghbi, H.Y., 2006. Enhanced anxiety stress-induced corticosterone release are associated with increased Crh expression in a mouse model of Rett syndrome. Proc. Natl. Acad. Sci. U.S.A. 103, 18267–18272. Mueller, B.R., Bale, T.L., 2008. Sex-specific programming of offspring emotionality after stress early in pregnancy. J. Neurosci. 28, 9055–9065. Murphy, D.L., Pigott, T.A., 1990. A comparative examination of a role for serotonin in obsessive compulsive disorder, panic disorder, and anxiety. J. Clin. Psychiatry 51, 53–60. Nishio, H., Kasuga, S., Ushijima, M., Harada, Y., 2001. Prenatal stress and postnatal development of neonatal rats—sex-dependent effects on emotional behavior and learning ability of neonatal rats. Int. J. Dev. Neurosci. 19, 37–45. Peters, D.A., 1982. Prenatal stress: effects on brain biogenic amine and plasma corticosterone levels. Pharmacol. Biochem. Behav. 17, 721–725. Peters, D.A., 1990. Maternal stress increases fetal brain and neonatal cerebral cortex 5-hydroxytryptamine synthesis in rats: a possible mechanism by which stress influences brain development. Pharmacol. Biochem. Behav. 35, 943–947. Rivera, H.M., Oberbeck, D.R., Kwon, B., Houpt, T.A., Eckel, L.A., 2009. Estradiol increases Pet-1 and serotonin transporter mRNA in the midbrain raphe nuclei of ovariectomized rats. Brain Res. 1259, 51–58. Sandford, J.J., Argyropoulos, S.V., Nutt, D.J., 2000. The psychobiology of anxiolytic drugs. Part 1: Basic neurobiology. Pharmacol. Ther. 88, 197–212. Seckl, J.R., Meaney, M.J., 2004. Glucocorticoid programming. Ann. N. Y. Acad. Sci. 1032, 63–84. Secoli, S.R., Teixeira, N.A., 1998. Chronic prenatal stress affects development and behavioral depression in rats. Stress 2, 273–280. Sodhi, M.S., Sanders-Bush, E., 2004. Serotonin and brain development. Int. Rev. Neurobiol. 59, 111–174. Szuran, T.F., Pliska, V., Pokorny, J., Welzl, H., 2000. Prenatal stress in rats: effects on plasma corticosterone, hippocampal glucocorticoid receptors, and maze performance. Physiol. Behav. 71, 353–362. Tamby Raja, R.L., Anderson, A.B., Turnbull, A.C., 1974. Endocrine changes in premature labour. Br. Med. J. 4, 67–71. Tan, H., Zhong, P., Yan, Z., 2004. Corticotropin-releasing factor and acute stress prolongs serotonergic regulation of GABA transmission in prefrontal cortical pyramidal neurons. J. Neurosci. 24, 5000–5008. Tsankova, N., Renthal, W., Kumar, A., Nestler, E.J., 2007. Epigenetic regulation in psychiatric disorders. Nat. Rev. Neurosci. 8, 355–367. Uphouse, L., Hiegel, C., Guptarak, J., Maswood, N., 2009. Progesterone reduces the effect of the serotonin 1B/1D receptor antagonist, GR127935, on lordosis behavior. Horm. Behav. 55, 169–174. Van den Hove, D.L., Steinbusch, H.W., Scheepens, A., Van de Berg, W.D., Kooiman, L.A., Boosten, B.J., Prickaerts, J., Blanco, C.E., 2005. Prenatal stress and neonatal rat brain development. Neuroscience 137, 145–155. Vallée, M., MacCari, S., Dellu, F., Simon, H., Le Moal, M., Mayo, W., 1999. Long-term effects of prenatal stress and postnatal handling on age-related glucocorticoid secretion and cognitive performance: a longitudinal study in the rat. Eur. J. Neurosci. 11, 2906–2916. Vallée, M., Mayo, W., Dellu, F., Le Moal, M., Simon, H., Maccari, S., 1997. Prenatal stress induces high anxiety and postnatal handling induces low anxiety in adult offspring: correlation with stress-induced corticosterone secretion. J. Neurosci. 17, 2626–2636. Valentino, R.J., Page, M.E., Curtis, A.L., 1991. Activation of noradrenergic locus coeruleus neurons by hemodynamic stress is due to local release of corticotropin-releasing factor. Brain Res. 555, 25–34. Walker, E., Mittal, V., Tessner, K., 2008. Stress and the hypothalamic pituitary adrenal axis activity in the developmental course of schizophrenia. Annu. Rev. Clin. Psychol. 4, 189–216. Walther, D.J., Bader, M., 2003. A unique central tryptophan hydroxylase isoform. Biochem. Pharmacol. 66, 1673–1680. Weaver, I.C., Cervoni, N., Champagne, F.A., D’Alessio, A.C., Sharma, S., Seckl, J.R., Dymov, S., Szyf, M., Meaney, M.J., 2004. Epigenetic programming by maternal behavior. Nat. Neurosci. 7, 847–854. Weinstock, M., 1997. Does prenatal stress impair coping and regulation of hypothalamic–pituitary–adrenal axis? Neurosci. Biobehav. Rev. 21, 1–10. Zimmerberg, B., Blaskey, L.G., 1998. Prenatal stress effects are partially ameliorated by prenatal administration of the neurosteroid allopregnanolone. Pharmacol. Biochem. Behav. 59, 819–827.
Contents lists available at ScienceDirect
Neuroscience Research journal homepage: www.elsevier.com/locate/neures
Rapid communication
Prenatal stress induces anxiety-like behavior together with the disruption of central serotonin neurons in mice Kazuya Miyagawa, Minoru Tsuji ∗ , Kanji Fujimori, Yasuho Saito, Hiroshi Takeda ∗∗ Division of Pharmacology, Department of Pharmaceutical Sciences, School of Pharmacy, International University of Health and Welfare, 2600-1 Kitakanamaru, Ohtawara, Tochigi 324-8501, Japan
a r t i c l e
i n f o
Article history: Received 28 October 2010 Received in revised form 19 January 2011 Accepted 7 February 2011 Available online 12 February 2011 Keywords: Serotonin Prenatal stress Neurodevelopment Anxiety Emotional behavior
a b s t r a c t Most pregnant women are at risk of showing some emotional abnormality, since some biological functions such as hormonal systems may dramatically change in pregnancy. Some of them may be exposed to strong stress as hesitation of positive drug therapies because of worries regarding adverse effects on the embryo. A growing body of evidence suggests that prenatal stress increases the vulnerability to neuropsychiatric disorders, including depression and anxiety. However, the mechanisms involved are still unknown. To clarify the influence of exposure to prenatal stress on emotional development, we examined behavioral responses in offspring exposed to weak- or strong-prenatal restraint stress. We found that offspring that had been exposed to strong stress displayed anxiety-like behavior as determined by the elevated plus-maze test. It has been widely accepted that central serotonin (5-hydroxytryptamine; 5-HT) neurons play a critical role in emotional behaviors. Immunohistochemical studies showed that exposure to strong-prenatal restraint stress increased the expression of 5-HT-positive cells in the dorsal raphe nuclei in mice. Moreover, under these conditions, tryptophan hydroxylase-like immunoreactivities were also dramatically increased. In contrast, these behavioral and neurochemical abnormalities were not observed in offspring that had been exposed to weak-prenatal restraint stress. These findings indicate that exposure to excessive prenatal stress induces anxiety-like behavior together with disruption of the development of 5-HT neurons in mice. © 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Depression and anxiety disorders are common public health problems with a lifetime prevalence of 10–20%, yet the mechanisms that underlie their pathophysiology are still poorly understood. Most pregnant women are at risk of showing some emotional abnormality, since some biological functions such as hormonal systems may dramatically change in pregnancy. Indeed, it has been reported that 16% of women experienced the onset of an affective disorder during pregnancy, and 68% of them showed symptoms during the first trimester (Kitamura et al., 1993). Thus, a suitable management plan that includes drug therapy may be necessary for pregnant women. However, doctors hesitate to use positive drug therapies because of worries regarding adverse effects on the embryo. Inappropriate mental support for pregnant women may result in their exposure to very stressful situations. Particularly when pregnant women suffer from serious mental disease, inappropriate drug therapies would expose them to the very severe
∗ Corresponding author. Tel.: +81 287 24 3489; fax: +81 287 24 3521. ∗∗ Corresponding author. Tel.: +81 287 24 3436; fax: +81 287 24 3521. E-mail addresses: [email protected] (M. Tsuji), [email protected] (H. Takeda).
stressful situations. Previous clinical research on pregnant women suffering depressive disorders indicated that the activity of the fetus and newborn is elevated, prenatal growth is delayed, and prematurity and low birthweight are more frequent (Field et al., 2006). Moreover, it has also been suggested that exposure to stress during gestation may impair the emotional development of the offspring and as a result the incidence of numerous neuropsychiatric disorders, including depression, anxiety, schizophrenia, and autism, may increase (Gillott and Standen, 2007; Koenig et al., 2002; Walker et al., 2008). Additionally, recent preclinical studies have suggested that prenatal stress induces several functional and structural abnormalities of the components that regulate stress responses, such as the hypothalamic–pituitary–adrenal (HPA)-axis (Lephart et al., 1997; Szuran et al., 2000) or monoamine neurotransmission (Peters, 1982, 1990; Weinstock, 1997). Moreover, the offspring exposed to prenatal stress show the abnormal psychiatric behaviors such as the increased fear and anxiety (Nishio et al., 2001; Vallée et al., 1997), persistent paradoxical sleep alterations (Dugovic et al., 1999), deficits of learning and memory (Hayashi et al., 1998; Vallée et al., 1999), depressive-like behavior (Secoli and Teixeira, 1998; Zimmerberg and Blaskey, 1998; Alonso et al., 1999) and schizophrenia-like behavior (Lehmann et al., 2000). However,
0168-0102/$ – see front matter © 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2011.02.002
112
K. Miyagawa et al. / Neuroscience Research 70 (2011) 111–117
the mechanisms accounting for the impact of the prenatal stress on the life of adulthood have not fully understood. On the other hand, axons from the neurons of the raphe nuclei, the principal source of serotonin (5-hydroxytryptamine; 5-HT) release in the brain, form a neurotransmitter system, reaching almost every part of the central nervous system. Recent clinical and preclinical studies have suggested that central 5-HT neurotransmission may be involved in the aetiology, expression and treatment of anxiety, impulsiveness and depression (Murphy and Pigott, 1990). In addition, it has been widely accepted that several medicines for psychiatric disorders were designed as mediator to 5-HT neurotransmission. 5-HT furthermore acts as a growth factor during embryogenesis, and serotonin receptor activity forms a crucial loop in the pathway leading to changes in brain structure (Sodhi and Sanders-Bush, 2004). Therefore, the incidence of numerous neuropsychiatric disorders caused by the exposure to stress during gestation might be associated with the disruption of 5-HT neurotransmission. Actually a few preclinical reports suggest that prenatal stress could affect on the central 5-HT neuron (Peters, 1982, 1990; Hayashi et al., 1998). On the basis of the previous reports, the aim of the present study was to clarify the influence of exposure to prenatal stress on the development of emotionality and central 5-HT neurons. The present studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the Committee on the Care and Use of Laboratory Animals of the International University Health and Welfare. All experiments were performed using 7–10-week-old male offspring of ICR mice (Japan SLC, Inc., Shizuoka, Japan) that had been prenatally exposed to stress as described below, or naive mice. The mice were housed at a room temperature of 23 ± 1 ◦ C and humidity of 50 ± 5% with a 12 h light–dark cycle (light on 7:00 a.m.–7:00 p.m.). Food and water were available ad libitum. Virgin female mice were mated at 10–11 weeks of age. The presence of a copulation plug denoted gestation day (GD) 0.5. Pregnant females were housed individually. In the present study, we used two types of prenatal stress model modified on the basis of previous reports (Chung et al., 2005; Van den Hove et al., 2005). In the model mice exposed to weak-prenatal restraint stress (WPRS), the pregnant mice were placed in a 50 (for mice with a body weight of 40 g or less)- or 70 (for mice with a body weight of 40 g or more)ml polystyrene tube three times a day (at approximately 10:00 a.m., 14:00 p.m., and 18:00 p.m.) for 1 h from GD 5.5 to GD 17.5. On the other hand, in the model mice exposed to strong-prenatal restraint stress (SPRS), the pregnant mice were placed in a tube as mentioned above at 6 h (10:00 a.m.–4:00 p.m.) a day. To investigate the changes in anxiety-like behaviors, 7–8 weeks old male offspring were tested using the elevated plus-maze paradigm (Muromachi Kikai Co., Ltd., Tokyo, Japan). The apparatus was elevated 40 cm from the ground and the maze consisted of two opposing open arms (30 cm × 6 cm × 0.3 cm) and two opposing enclosed arms (30 cm × 6 cm × 15 cm) that were connected by a central platform (6 cm × 6 cm, 40 lux), thus forming the shape of a plus sign. Each animal was placed on a central platform, and the distance that the mouse moved in the maze was recorded for 5 min by an overhead color CCD camera that tracked the centre of the mouse. Moreover, the time spent in and the numbers of entry into open or enclosed arms were also recorded. Data from the CCD camera were collected through a custom-designed interface (CAT-10; Muromachi Kikai) as a reflection signal. All of the data were analyzed and stored in a personal computer using analytical software (Comp ACT HBS, Muromachi Kikai). The results were calculated as mean ratios of the time spent in the open arms to the total time spent in both the open and enclosed arms. Entries into the open arms (%), total entries, moving distance (cm) and moving speed (cm/s) were also scored.
After elevated plus-maze paradigm, the mice were immediately placed in the center of open field (Muromachi Kikai) and the behavior of the mice in the open field was recorded for 5 min. The apparatus of open field paradigm was made of gray wooden box (50 cm × 50 cm × 50 cm, 170 lux). An infrared beam sensor was installed on the wall to detect the numbers and duration of rearing behaviors. Other behavioral performance, such as locus and the distance of movement (total locomotor activity (cm)) of mice, was recorded by an overhead color CCD camera. The head of the mice were painted yellow and the color CCD camera followed the center of gravity. Data from the CCD camera were collected through a custom-designed interface (CAT-10; Muromachi Kikai) as a reflection signal. All of the data were analyzed and stored in a personal computer using analytical software (Comp ACT HBS, Muromachi Kikai). The results were calculated as thigmotaxis (amount of time in the outer versus inner zone (33 cm × 33 cm)). The other parameters such as rearing behavior (count and duration), moving distance and moving speed (cm/s) were also scored. In the immunohistochemical analysis, mice were deeply anesthetized with sodium pentobarbital (70 mg/kg, i.p.) and perfusion-fixed with 4% paraformaldehyde (Wako Pure Chemical Industries Ltd., Osaka, Japan) in PBS. The brains were post-fixed in 4% paraformaldehyde in PBS for 12 h at 4 ◦ C. Brain sections (80 m thick) were prepared on a Microslicer (DTK-1000; Ted Pella, Inc., CA). The sections were incubated with 10% normal goat serum in ice-cold PBS for 60 min to block nonspecific antibody binding and then with the 5-HT rabbit monoclonal antibody (1:1000 dilution; Sigma Chemical, St. Louis, MO) or tryptophan hydroxylase (TPH) mouse monoclonal antibody (1:600; Sigma Chemical) for 2 days at 4 ◦ C. The samples were then rinsed with PBS and incubated with the appropriate secondary antibody conjugated with Alexa 488 (1:1000) for 12 h at 4 ◦ C. The sections were then rinsed with PBS and mounted on glass slides with 20% glycerol in PBS. Fluorescence immunolabeling was detected using a confocal laserscanning microscope (FV1000; Olympus Optical, Tokyo, Japan). The intensity of TPH-like immunoreactivity in dorsal raphe nuclei (DRN) was analyzed and quantified by computer-assisted densitometry using Image J software (National Institutes of Health, MD). The data are expressed as the mean with SEM. Statistical analyses were performed using Student’s t-test. The influence of prenatal exposure to stress on anxiety-like behaviors in mice was evaluated by the elevated plus-maze test. The results in mice that had been exposed to prenatal restraint stress are shown in Fig. 1. As shown in Fig. 1A and B, mice that had been exposed to WPRS show hypo-locomotion in the elevated plus-maze (Fig. 1A (moving distance); **p < 0.01 vs. control, Fig. 1B (number of total entries); **p < 0.01 vs. control). Under these conditions, WPRS-exposed mice failed to show the change in the percentage of entries into open arms (Fig. 1C) as well as percentage of time spent in open arms (Fig. 1D). Like WPRS-exposed mice, mice that had been exposed to SPRS show hypo-locomotion in the elevated plus-maze (Fig. 1F (moving distance); *p < 0.05 vs. control, Fig. 1G (number of total entries); ***p < 0.001 vs. control). On the other hand, the percentage of entries into open arms (Fig. 1H; **p < 0.01 vs. control) and the percentage of time spent in open arms (Fig. 1I; *p < 0.01 vs. control) were significantly decreased in SPRS-exposed mice. As another measurement of emotionality, the mice that had been prenatally exposed to restraint stress were evaluated by the open field test (Fig. 2). As shown in Fig. 2A–D, WPRS-exposed mice failed to show change in the thigmotaxis and general activity levels (total distance traveled and rearing) in open field. On the other hand, although SPRS-exposed mice failed to show change in the thigmotaxis (Fig. 2F), rearing count (Fig. 2G; **p < 0.01 vs. control), rearing duration (Fig. 2H; ***p < 0.001 vs. control) and moving distance (Fig. 2I; *p < 0.05 vs. control) were significantly
K. Miyagawa et al. / Neuroscience Research 70 (2011) 111–117
113
Fig. 1. Anxiety-like behaviors of mice that had been prenatally exposed to restraint stress as detected by the elevated plus-maze test. (A–E) Analysis in the WPRS-exposed mice. (F–J) Analysis in the SPRS-exposed mice. Each mouse placed on a central platform, and the distance that the mouse moved in the maze was recorded for 5 min. Moreover, the moving distance (A, F), the numbers of total entries into open and enclosed arms (B, G), the percentage of entries into open arms (C, H), percentage of time spent in the open arms (D, I) and moving speed (E, J) were scored. The representative images showing the mouse traveling patterns are shown in panel I. Each column represents the mean with SEM of 13-20 mice. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. Cont: control, WPRS: weak prenatal restraint stress, SPRS: strong prenatal restraint stress.
114
K. Miyagawa et al. / Neuroscience Research 70 (2011) 111–117
Fig. 2. General behaviors of mice that had been prenatally exposed to restraint stress as detected by the open field procedure. (A–E) Analysis in the WPRS-exposed mice. (F–J) Analysis in the SPRS-exposed mice. Each mouse was placed in the center of open field and the behavior of the mouse in the open field was recorded for 5 min. Moreover, the percentage of time spent in central area (A, F), the rearing counts (B, G) and duration (C, H), the moving distance (D, I) and moving speed (E, J) were scored. The representative images showing the mouse traveling patterns are shown in panel I. Rearing behaviors are represented as closed diamonds. Each column represents the mean with SEM of 13-20 mice. *p < 0.05, ***p < 0.001 vs. control. Cont: control, WPRS: weak prenatal restraint stress, SPRS: strong prenatal restraint stress.
K. Miyagawa et al. / Neuroscience Research 70 (2011) 111–117
decreased. These results indicate that exposure to excessive prenatal stress could induce the emotional abnormality, i.e., increase in anxiety levels in offspring. These results of behavioral studies suggest that SPRS-exposed mice could reflect the clinical case that the severe stressful situations during pregnancy impair the emotional development of offspring. The hypo-locomotion in the behavioral tests could be also observed if the mice exposed to prenatal stress presented motor dysfunction including delayed growth. In the present study, although we have not checked the motor functions using rota-rod test and so on, WPRS- and/or SPRS-exposed mice failed to show a clear motor deficit in their home cage and behavioral experiment apparatuses. Actually, the moving speeds were not significantly different in both experiment and in both group (Fig. 1Eand J, Fig. 2Eand J). Therefore, the hypo-locomotion observed in the present behavioral tests could indicate the emotional dysfunction induced by the exposure to prenatal restraint stress. The reason of why the hypo-locomotion of WPRS group was only observed by elevated plus-maze test could not be clearly explained. In our opinion, the fear level in the elevated plus maze paradigm is higher than that in the open field paradigm. Therefore, the present data suggest that although WPRS-exposed mice could show the abnormal emotional behavior in only powerful fear condition, SPRS-exposed mice could also show in the mild fear condition. Additionally, the reason of why that SPRS-exposed mice failed to show decrease in percent of time spent in central area in the open-field test also could not be clearly explained. One possible explanation is that the decrease in time spent in open area is observed only under the extreme stress condition, such as acute stress stimuli. Growing body of evidence suggests that brain 5-HT is implicated in the etiology of neuropsychiatric disorders, such as anxiety and depression (Murphy and Pigott, 1990). Most antidepressants or anxiolytics change the levels of 5-HT in the brain (Krishnan and Nestler, 2008). This suggests that biochemical imbalances within the central 5-HT systems may underlie the pathogenesis of these disorders. The DRN, a major source of 5-HT innervation of the forebrain, plays a critical role in stress responsiveness and the effects of antidepressant drugs commonly used to treat anxiety disorders (Sandford et al., 2000). Therefore, we next investigated the changes in 5-HT neurons in DRN of mice that had been prenatally exposed to restraint stress (Fig. 3). An immunohistochemical study showed that exposed to WPRS failed to change in the expression of 5-HTpositive cells (Fig. 3B–D). On the other hand, SPRS dramatically increased the expression of 5-HT-positive cells in DRN compared to the control (Fig. 3E–G; *p < 0.05 vs. control). To our knowledge, this is the first evidence that prenatal stress could induce structural abnormalities in the development of 5-HT neurons in DRN. We then investigated whether 5-HT neurons were functionally overexpressed in DRN of mice that had been prenatally exposed to restraint stress. Similar to 5-HT-positive cells, TPH-like immunoreactivities in DRN were also dramatically increased in SPRS-exposed mice compared with the control (Fig. 3K–M; *p < 0.05 vs. control), although such a neurochemical changes were not observed in WPRS-exposed mice (Fig. 3H–J). It has been widely accepted that TPH is the first-step and rate-limiting enzyme involved in the synthesis of 5-HT in the brain (Walther and Bader, 2003). Taken together, these results indicate that excessive prenatal restraint stress induces the overexpression of functional 5-HT neurons in DRN in mice. As mentioned above, a few preclinical reports suggest that prenatal stress could affect on the central 5-HT neurons. Peters (1990) reported that maternal stress increased fetal brain 5-HT synthesis in rat. They also found that the offspring exposed to prenatal stress showed region-specific changes in brain 5-HT, 5hydroxyindoleacetic acid (5-HIAA) and norepinephrine levels in infancy (Peters, 1982). Hayashi et al. (1998) reported that mater-
115
nal stress induced synaptic loss associated with disruption of 5-HT neurotransmission and developmental disabilities of offspring. In addition to these precedent articles, we here report a novel finding that the 5-HT positive neurons significantly increase in DRN of prenatal-stress mice. Although it has been widely accepted that brain 5-HT neuron play an important role in emotional behavior, its clear mechanism is not understood. There are various theories linking the function of 5-HT and its receptors to the actions of both anxiolytic and antidepressant drugs. As known in serotonin hypothesis, pharmacological manipulation to enhance 5-HT concentration in the brain increases anxiety (Handley and McBlane, 1993), in contrast, a reduction in 5-HT concentrations is associated with reduced anxiety. This suggests that anxiety is caused by abnormal increase in brain 5-HT concentrations. The present findings therefore suggest that prenatal stress may induces the superabundant brain 5-HT levels by overdevelopment of 5-HT neuronal cells, as a result, anxiety-like behavior in enhanced. Further studies should be needed to prove this hypothesis. Although the present findings do not fully explain the underlying mechanisms, they are supported by some recent preclinical studies. One of the potential mechanism by which stress can influence fetal development is the disturbance of hormonal systems in developmental stage. Especially, plasma estrogen and progesterone large increase during pregnancy (Tamby Raja et al., 1974). Moreover, prenatal stress could disrupt these hormonal systems (Field et al., 2006). To our knowledge the direct evidence that prenatal stress could affect 5-HT neurodevelopment via the disruption of these hormonal systems has not been reported. On the other hand, it has been recently indicated that these sex hormones interact with the 5-HT neuronal systems (Rivera et al., 2009; Uphouse et al., 2009). Additionally, the gender difference in 5-HT neurotransmission has also been reported (Kakeyama et al., 2002; Jovanovic et al., 2008). It is possible that sex hormones might be involved in mechanisms of present findings. The other potential mechanism is epigenetics, including DNA methylation (Seckl and Meaney, 2004; Tsankova et al., 2007; Weaver et al., 2004). Furthermore, attention has recently been focused on the involvement of epigenetic machinery in the programming of early life events. corticotropin releasing factor (CRF) and the glucocorticoid receptor (GR), which are important components involved in the stress response of the body, have both been previously shown to be regulated by methylation and are programmed early in life (Tsankova et al., 2007; Weaver et al., 2004). CRF is a known regulator of 5-HT neurotransmission (Bale and Vale, 2004; McGill et al., 2006; Tan et al., 2004; Valentino et al., 1991). During pregnancy, both plasma CRF and corticosteroid increase (Kammerer et al., 2006). Therefore, the dysregulation of CRF could be an upstream contributor to abnormalities in 5-HT transmission and a stress-sensitive phenotype. In fact, Mueller and Bale (2008) recently reported that changes in CRF and GR gene methylation correlated with altered gene expression, and thus provided important evidence of abnormal epigenetic programming during early prenatal stress. Therefore, it is expected that such epigenetic factors might play a role in the present findings. Furthermore, we should focus on the changes in functional molecules involved in the development of 5-HT neurons. 5-HT is an important neurotransmitter that has multiple functions in the central nervous system. Its synthesis is restricted to a very limited number of cells in the brainstem raphe nuclei with a vast axonal network. Alenia et al. (2006) reported that these cells express several markers of the serotonin lineage such as the rate-limiting enzyme in serotonin synthesis TPH2, the serotonin transporter, and the transcription factor Pet1. Pet1, LIM homeobox transcription factor 1b (Lmx1b), NK transcription factor related, locus 2 (Nkx2.2), Mouse achaete-scute homolog 1
116
K. Miyagawa et al. / Neuroscience Research 70 (2011) 111–117
Fig. 3. Morphological changes in 5-HT neurons in DRN of male mice that had been prenatally exposed to restraint stress. (A) Schematic diagram of the range of DRN enclosed by the line. (B–G) Typical photographs of 5-HT-positive cells in the DRN of control (B, E), WPRS-exposed (C) and SPRS-exposed (F) mice. The results of a quantitative analysis of 5-HT-positive cells in the DRN are shown in panels D and G. (H–M) Typical photographs of TPH-like immunoreactivity (IR) in the DRN of control (H, K), WPRS-exposed (I) and SPRS-exposed (L) mice. The results of a quantitative analysis of TPH-IR in the DRN are shown in panel J and M. The column represents the mean with SEM of 4–6 independent samples. *p < 0.05 vs. control mice. Scale bars: 200 m. Cont: control, WPRS: weak prenatal restraint stress, SPRS: strong prenatal restraint stress.
K. Miyagawa et al. / Neuroscience Research 70 (2011) 111–117
(Mash1), GATA-motif binding (Gata) 2, Gata3, and Paired-like homeodomain protein 2b (Phox2b) form a transcriptional network, which specifies the differentiation of serotonergic neurons around embryonic day 11 in the mouse. Recently, we found that prenatal stress also induces up-regulation of 5-HT-positive cells in the embryonic brain of mice (unpublished observation). Therefore, it is expected that stress stimuli could disrupt the transcriptional networks of 5-HT neurons across gestation. Further studies will be necessary to clarify the effects of prenatal stress on the development of 5-HT neurons in the embryonic brain. In summary, we found that excessive prenatal stress induced long-term emotional abnormalities associated with morphological and/or functional changes in central 5-HT neurons in mice. Furthermore, the disruption of 5-HT neuron development seems to also be induced by exposure to stress across gestation. The present findings provide evidence that prenatal stress is an important risk factor that underlies the induction of neuropsychiatric disorders in adulthood. References Alenia, N., Bashammakh, S., Bader, M., 2006. Specification and differentiation of serotonergic neurons. Stem Cell Rev. 2, 5–10. Alonso, S.J., Castellano, M.A., Quintero, M., Navarro, E., 1999. Action of antidepressant drugs on maternal stress-induced hypoactivity in female rats. Methods Find. Exp. Clin. Pharmacol. 21, 291–295. Bale, T.L., Vale, W.W., 2004. CRF and CRF receptors: role in stress responsivity and other behaviors. Annu. Rev. Pharmacol. Toxicol. 44, 525–557. Chung, S., Son, G.H., Park, S.H., Park, E., Lee, K.H., Geum, D., Kim, K., 2005. Differential adaptive responses to chronic stress of maternally stressed male mice offspring. Endocrinology 146, 3202–3210. Dugovic, C., Maccari, S., Weibel, L., Turek, F.W., Van Reeth, O., 1999. High corticosterone levels in prenatally stressed rats predict persistent paradoxical sleep alterations. J. Neurosci. 19, 8656–8664. Field, T., Diego, M., Hernandez-Reif, M., 2006. Prenatal depression effects on the fetus and newborn: a review. Infant. Behav. Dev. 29, 445–455. Gillott, A., Standen, P.J., 2007. Levels of anxiety and sources of stress in adults with autism. J. Intellect. Disabil. 11, 359–370. Handley, S.L., McBlane, 1993. 5HT drugs in animal models of anxiety. Psychopharmacology 112, 13–20. Hayashi, A., Nagaoka, M., Yamada, K., Ichitani, Y., Miake, Y., Okado, N., 1998. Maternal stress induces synaptic loss and developmental disabilities of offspring. Int. J. Dev. Neurosci. 16, 209–216. Jovanovic, H., Lundberg, J., Karlsson, P., Cerin, A., Saijo, T., Varrone, A., Halldin, C., Nordström, A.L., 2008. Sex differences in the serotonin 1A receptor and serotonin transporter binding in the human brain measured by PET. Neuroimage 39, 1408–1419. Kakeyama, M., Umino, A., Nishikawa, T., Yamanouchi, K., 2002. Decrease of serotonin metabolite in the forebrain facilitation of lordosis by dorsal raphe nucleus lesions in male rats. Endocr. J. 49, 573–579. Kammerer, M., Taylor, A., Glover, V., The HPA, 2006. axis and perinatal depression: a hypothesis. Arch. Womens Ment. Health. 9, 187–196. Kitamura, T., Shima, S., Sugawara, M., Toda, M.A., 1993. Psychological and social correlates of the onset of affective disorders among pregnant women. Psychol. Med. 23, 967–975. Koenig, J.I., Kirkpatrick, B., Lee, P., 2002. Glucocorticoid hormones and early brain development in schizophrenia. Neuropsychopharmacology 24, 309–318. Krishnan, V., Nestler, E.J., 2008. The molecular neurobiology of depression. Nature 455, 894–902. Lehmann, J., Stöhr, T., Feldon, J., 2000. Long-term effects of prenatal stress experiences and postnatal maternal separation on emotionality and attentional processes. Behav. Brain. Res. 107, 133–144. Lephart, E.D., Watson, M.A., Jacobson, N.A., Rhees, R.W., Ladle, D.R., 1997. CalbindinD28k is regulated by adrenal steroids in hypothalamic tissue during prenatal development. Brain. Res. Dev. Brain. Res. 100, 117–120.
117
McGill, B.E., Bundle, S.F., Yaylaoglu, M.B., Carson, J.P., Thaller, C., Zoghbi, H.Y., 2006. Enhanced anxiety stress-induced corticosterone release are associated with increased Crh expression in a mouse model of Rett syndrome. Proc. Natl. Acad. Sci. U.S.A. 103, 18267–18272. Mueller, B.R., Bale, T.L., 2008. Sex-specific programming of offspring emotionality after stress early in pregnancy. J. Neurosci. 28, 9055–9065. Murphy, D.L., Pigott, T.A., 1990. A comparative examination of a role for serotonin in obsessive compulsive disorder, panic disorder, and anxiety. J. Clin. Psychiatry 51, 53–60. Nishio, H., Kasuga, S., Ushijima, M., Harada, Y., 2001. Prenatal stress and postnatal development of neonatal rats—sex-dependent effects on emotional behavior and learning ability of neonatal rats. Int. J. Dev. Neurosci. 19, 37–45. Peters, D.A., 1982. Prenatal stress: effects on brain biogenic amine and plasma corticosterone levels. Pharmacol. Biochem. Behav. 17, 721–725. Peters, D.A., 1990. Maternal stress increases fetal brain and neonatal cerebral cortex 5-hydroxytryptamine synthesis in rats: a possible mechanism by which stress influences brain development. Pharmacol. Biochem. Behav. 35, 943–947. Rivera, H.M., Oberbeck, D.R., Kwon, B., Houpt, T.A., Eckel, L.A., 2009. Estradiol increases Pet-1 and serotonin transporter mRNA in the midbrain raphe nuclei of ovariectomized rats. Brain Res. 1259, 51–58. Sandford, J.J., Argyropoulos, S.V., Nutt, D.J., 2000. The psychobiology of anxiolytic drugs. Part 1: Basic neurobiology. Pharmacol. Ther. 88, 197–212. Seckl, J.R., Meaney, M.J., 2004. Glucocorticoid programming. Ann. N. Y. Acad. Sci. 1032, 63–84. Secoli, S.R., Teixeira, N.A., 1998. Chronic prenatal stress affects development and behavioral depression in rats. Stress 2, 273–280. Sodhi, M.S., Sanders-Bush, E., 2004. Serotonin and brain development. Int. Rev. Neurobiol. 59, 111–174. Szuran, T.F., Pliska, V., Pokorny, J., Welzl, H., 2000. Prenatal stress in rats: effects on plasma corticosterone, hippocampal glucocorticoid receptors, and maze performance. Physiol. Behav. 71, 353–362. Tamby Raja, R.L., Anderson, A.B., Turnbull, A.C., 1974. Endocrine changes in premature labour. Br. Med. J. 4, 67–71. Tan, H., Zhong, P., Yan, Z., 2004. Corticotropin-releasing factor and acute stress prolongs serotonergic regulation of GABA transmission in prefrontal cortical pyramidal neurons. J. Neurosci. 24, 5000–5008. Tsankova, N., Renthal, W., Kumar, A., Nestler, E.J., 2007. Epigenetic regulation in psychiatric disorders. Nat. Rev. Neurosci. 8, 355–367. Uphouse, L., Hiegel, C., Guptarak, J., Maswood, N., 2009. Progesterone reduces the effect of the serotonin 1B/1D receptor antagonist, GR127935, on lordosis behavior. Horm. Behav. 55, 169–174. Van den Hove, D.L., Steinbusch, H.W., Scheepens, A., Van de Berg, W.D., Kooiman, L.A., Boosten, B.J., Prickaerts, J., Blanco, C.E., 2005. Prenatal stress and neonatal rat brain development. Neuroscience 137, 145–155. Vallée, M., MacCari, S., Dellu, F., Simon, H., Le Moal, M., Mayo, W., 1999. Long-term effects of prenatal stress and postnatal handling on age-related glucocorticoid secretion and cognitive performance: a longitudinal study in the rat. Eur. J. Neurosci. 11, 2906–2916. Vallée, M., Mayo, W., Dellu, F., Le Moal, M., Simon, H., Maccari, S., 1997. Prenatal stress induces high anxiety and postnatal handling induces low anxiety in adult offspring: correlation with stress-induced corticosterone secretion. J. Neurosci. 17, 2626–2636. Valentino, R.J., Page, M.E., Curtis, A.L., 1991. Activation of noradrenergic locus coeruleus neurons by hemodynamic stress is due to local release of corticotropin-releasing factor. Brain Res. 555, 25–34. Walker, E., Mittal, V., Tessner, K., 2008. Stress and the hypothalamic pituitary adrenal axis activity in the developmental course of schizophrenia. Annu. Rev. Clin. Psychol. 4, 189–216. Walther, D.J., Bader, M., 2003. A unique central tryptophan hydroxylase isoform. Biochem. Pharmacol. 66, 1673–1680. Weaver, I.C., Cervoni, N., Champagne, F.A., D’Alessio, A.C., Sharma, S., Seckl, J.R., Dymov, S., Szyf, M., Meaney, M.J., 2004. Epigenetic programming by maternal behavior. Nat. Neurosci. 7, 847–854. Weinstock, M., 1997. Does prenatal stress impair coping and regulation of hypothalamic–pituitary–adrenal axis? Neurosci. Biobehav. Rev. 21, 1–10. Zimmerberg, B., Blaskey, L.G., 1998. Prenatal stress effects are partially ameliorated by prenatal administration of the neurosteroid allopregnanolone. Pharmacol. Biochem. Behav. 59, 819–827.