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The effects of prenatal stress on expression of p38 MAPK in offspring hippocampus
Int. J. Devl Neuroscience 26 (2008) 535–540 www.elsevier.com/locate/ijdevneu
The effects of prenatal stress on expression of p38 MAPK in offspring hippocampus Qing Cai a, Shuyun Huang a, Zhongliang Zhu b,c,*, Hui Li d, Qinghong Li d, Ning Jia c, Jankang Liu e,f a Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China College of Life Science, Northwest University, Xi’an, Shaan xi 710069, PR China c Department of Physiology and Pathophysiology, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaan xi 710061, PR China d Department of Pediatrics, Xi’an Jiaotong University First Hospital, Xi’an, PR China e Institute for Nutritional Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China f Institute for Brain Aging, University of California, Irvine, CA 92796, United States b
Received 27 April 2008; received in revised form 3 June 2008; accepted 3 June 2008
Abstract The hippocampus, which has the highest density of GC receptors in the brain, is involved in the regulation of the HPA and the behavioral responses to stress. Overexposure to corticosteroid hormones is harmful to hippocampal neuron integrity. Our purpose is to investigate the effects of prenatal stress (PNS) on expression of p38 mitogen-activated protein kinase (p38 MAPK) in offspring hippocampal neurons using Western blotting and Immunohistochemistry. The prenatal restraint stress induces significant increase in the expression of p-p38 MAPK and total p38 MAPK in female offspring hippocampus. The level of p-p38 MAPK in PNS female offspring rats was significantly increased (126.41 3.937, n = 6) compared with that in the control female offspring rats (101.35 3.468, n = 6, P < 0.01). Immunoblot analysis revealed there was significant difference in the level of total p38 MAPK between the female control and prenatal restraint stress offspring rats (101.70 3.162 vs. 128.111 2.724, respectively, P < 0.01). Immunodensity of p38 MAPK was significantly increased above female control in PNS female offspring hippocampal CA3 and CA4 fields (P < 0.001 vs. control group, CON). The data suggest that exposure of animals to a period of stressful experience during a critical phase could impose lasting effects on the offspring hippocampal neurons cellular signalling of offspring hippocampus. Crown Copyright # 2008 Published by Elsevier Ltd on behalf of ISDN. All rights reserved. Keywords: Prenatal stress; p38 MAPK; Hippocampus; CA3
1. Introduction Numerous studies show that stressors acting upon the organism during pregnancy can have distinct and long lasting effects on the offspring. Prenatally stressed offspring may exhibit alterations in brain morphology and behavior (Pivina et al., 2007; Lee et al., 2007), increased emotional reactivity (Bosch et al., 2007; Cannizzaro et al., 2008) and altered regulation of the hypothalamic–pituitary–adrenal (HPA) axis (Weinberg et al., 2008). It was reported hyperactivity of HPA axis in offspring induced by prenatal stress (PNS) was related to the high levels of maternal corticosterone secretion during
* Corresponding author at: School of Medicine, Xi’an Jiaotong University, Xi’an 710061, PR China. Tel.: +86 29 82655 669. E-mail address: [email protected] (Z. Zhu).
restraint stress (Maccari et al., 2003). The hippocampus, which has the highest density of glucocorticoid (GC) receptors in the brain, is involved in the regulation of the HPA and the behavioral responses to stress. A previous study reported that levels of phosphorylated mitogen-activated protein kinase and p38 MAPK, as well as phosphorylated calcium/calmodulindependent protein kinase II were enhanced shortly after swim stress (Ahmed et al., 2006). We have investigated the effects of PNS on the intracellular Ca2+ and reactive oxygen species (ROS) in hippocampal CA3 region of female offspring. PNS caused an increase in the concentrations of the intracellular calcium and the production of ROS in hippocampal CA3 region (Zhu et al., 2004). Our previous studies have demonstrated that PNS can increase the expression of p-ERK2 compared to that in the control female offspring rats and total ERK2 in female offspring hippocampus. ERK immunodensity was significantly increased in PNS groups
0736-5748/$34.00. Crown Copyright # 2008 Published by Elsevier Ltd on behalf of ISDN. All rights reserved. doi:10.1016/j.ijdevneu.2008.06.001
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Q. Cai et al. / Int. J. Devl Neuroscience 26 (2008) 535–540
in CA3 field in male offspring hippocampus compared with control animals. ERK optical density was significantly increased in PNS female offspring hippocampus CA1, CA3 and CA4 region (Cai et al., 2007a,b). Whether PNS causes the changes of p38 MAPK in offspring hippocampus neurons is unknown. In the present study Western blotting were employed to examine the effects of PNS on expression of p38 MAPK in offspring hippocampal neurons.
in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) for 1 h at room temperature and incubated with the primary antibody in TBSTwith 5% free-fat dry milk overnight at 4 8C with gentle shaking. The membranes were rinsed with TBST three times. The p38 MAPK levels were evaluated respectively with rabbit polyclonal antibodies that recognize unphosphorylated and phosphorylated p38 MAPK (1:1000 dilutions, Cell Signalling, Beverly, MA, USA) for 1 h at room temperature. Reactive proteins were detected with an ECL Western Blotting Detection Kit (AP Biotech, Buckinghamshire, UK). Protein loading was assessed by re-probing the blots with mouse anti-rat b-actin antibody.
2.3. Immunohistochemistry 2. Materials and methods 2.1. Animals and procedures All procedures were carried out in accordance with the United States Public Health Services Guide for care and use of laboratory animals and were approved by the Institutional Animals Care and Use Committee at Xi’an Jiaotong University. All efforts were made to minimize the number of animals used and their suffering. A total of 15 female and 5 male Sprague–Dawley rats were adopted. Female rats weighting 230–250 g and male rats weighting 280–350 g were used. Animals were provided by the experimental animal center of Xi’an Jiaotong University Medical College. The rats were housed in an animal room with controlled temperature (22 2 8C) and humidity (60%) controlled animal room on a 12 h light/dark cycle (light on from 8:00 a.m. to 8:00 p.m.) with free access to food and water throughout the experiment. Virgin female rats were placed overnight with adult male rats (3:1) for mating. Vaginal smear was examined on the following morning. The day on which the smear was spermpositive was determined as embryonic day 0. Each pregnant rat was then housed separately. A total of 12 pregnant rats were randomly assigned to prenatal stress (n = 6) and control groups (n = 6). The pregnant rats of PNS group were exposed to restraint stress on days 14–20 of pregnancy three times daily for 45 min (Koehl et al., 1999; McCormick et al., 1995). To prevent habituation of animals to the daily procedure, restraint periods were randomly shifted within certain time periods (08:00–11:00 a.m., 11:00 a.m.–02:00 p.m., and 04:00–07:00 p.m.). The restraint device was a transparent plastic tube (6.8 cm in diameter) with air holes for breathing and closed end. The length could be adjusted to accommodate the size of the animals. After birth, offspring of all groups were housed in the same animal room, and kept together with their biologic mothers. The pregnant rats of the control group were left undisturbed. On day 21, after all offspring were weaned, male and female pups were separated and housed four in each cage, respectively until testing at 1 month of age. At the end of postnatal day 30, one male and female offspring rat from the same dam have been selected with a random choice in two experiments. Six male and six female rats were used for Western blotting and Immunohistochemistry, respectively. The light phases in which data was collected.
2.2. Gel electrophoresis and Western blotting Six male and six female rats from different dams were decapitated under deep anesthesia with chloral hydrate (400 mg/kg, i.p.). The hippocampus was dissected out on the petri dish filled with ice and immediately frozen in liquid nitrogen and then kept in a 80 8C freezer until later experimental use. Each hippocampus was homogenized with a glass tissue grinder in 500 ml cold phosphate-buffered saline buffer containing protease inhibitors: 2 mg/mL aprotinine, 1 mg/mL leupeptine, and 1 mmol/L PMSF (St. Louis, MO, USA). The samples were centrifuged at 12,000 g for 20 min at 4 8C after homogenization. The total protein concentration in each sample was determined using the BCA protein assay reagent kit (Pierce, Rockford, IL, USA). The sample proteins (50 mg per lane) were separated by electrophoresis performed on 10% sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE, Bio-Rad, Barcelona, Spain) at room temperature. Following electrophoresis, the proteins in the gel were transferred to 0.45 mm polyvinyldifluoride membranes (PVDF). The membrane was blocked with 5% free-fat dry milk
Under deep anesthesia with chloral hydrate, the rats of each group were perfused transcardially with approximately 200 mL 0.1 mol/L PBS (pH 7.4), followed by approximately cold 400 mL 4% paraformaldehyde in PB (pH 7.4). The brains were removed, cut into blocks containing the hippocampus and post-fixed in the same solution for 24 h at 4 8C. Subsequently, the tissue blocks were dehydrated, processed for paraffin embedding and then cut into frontal sections 4 mm thickness. Brain sections were deparaffinized in xylene and hydrated through graded alcohols to Tris-buffered saline, washed with TBS three times (5 min each time). They were then treated with freshly made 3% H2O2 in TBS for 30 min. After washed several times in TBS, they were then treated with 0.1% Triton X-100. Nonspecific binding sites were blocked by incubation with normal goat serum 1 h at 37 8C. The sections were then incubated with polyclonal IgG anti-p38 MAPK (1:200 dilutions, Santa Cruz Biotechnology, CA) at 4 8C overnight. Biotinylated goat antirabbit IgG (1:500 dilutions, Beijing ZhongShan Biotechnology, China) used as the secondary antibody was incubated with the sections for 1 h at room temperature and detected with the streptavidin–peroxidase complex. The immunoreaction was visualized by treating the sections with DAB kit at room temperature. The sections were then dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped. Immunohistochemical controls were performed using the same procedures as described above except that the primary antibodies were omitted. No positive immunostaining was found in any controls (not shown). For qualitative evaluations of p38 MAPK immunoreactivity, the number of p38 MAPK and immunoreactive cells was counted using a computer-based imaging system (AnaliSYS, Soft Imaging System GmbH, Munster, Germany) with the images captured using a digital camera. The optical measurements were made blindly to the animal groups. Optical density (OD) was measured for estimation of quantitative differences in p38 MAPK immunoreactivity between experimental groups. OD analysis was performed on high resolution and analyzed with Scion Image Software based on NIH image (NIH Image, Bethesda, MD, USA). Background values were obtained from the neighboring white matter. The average of values from three sections for each subject was used for statistical analysis.
2.4. Statistics All values reported are mean S.E.M. Data analyses were performed using the software of SPSS 11.0. Two-way (prenatal treatment-gender) analysis of variance (ANOVA) was used to examine differences between male and female offspring and among the groups, and the interaction between group and gender. A difference was considered significant at P < 0.05 level.
3. Results 3.1. Effects of PNS on expression of phosphorylated p38 MAPK and total p38 MAPK in offspring hippocampus Phosphorylated p38 MAPK activation was analyzed using specific antibodies, by Western blotting from total hippocampal extracts. The results of Western blotting were shown in Fig. 1.
Q. Cai et al. / Int. J. Devl Neuroscience 26 (2008) 535–540
Fig. 1. (A) Effects of PNS on expression of total p38 MAPK in offspring hippocampus. Representative immunoblot of p38 MAPK (top lane). Cytosolic fractions (50 mg protein/lane) from CON male (lane 1), CON female (lane 2), PNS male (lane 3) and PNS female offspring hippocampus (lane 4) were subjected to 10% SDS–PAGE and Western blot analysis using an anti p38 MAPK antibody. Representative immunoblot for b-actin to control protein loading (low lane). (B). Summary of results of total p38 MAPK levels in the hippocampus. Quantitative data based on the measurement of integrated optical density in Western blot illustrated in (A). The levels of total p38 MAPK protein were significantly higher in PNS female offspring hippocampus than that of control female offspring hippocampus (P < 0.01).
Each value was expressed relative to the mean density of the control group male rat. Mean density of protein bands in the control group was expressed as 100%. There were no differences of mean density of protein between the control group male and female rats. Immunoblot analysis of total p38 MAPK revealed that there was no significant difference in the level of p38 MAPK between the male control and PNS male offspring rats and control female offspring rats (100.57 3.409, 102.08 4.450, n = 6 for each group). Immunoblot analysis revealed there was significant difference in the level of total p38 MAPK between the female control and PNS offspring rats (101.70 3.162, 128.111 2.724, respectively, F = 12.547, P < 0.01) (Fig. 1A and B). Quantitative Western immunoblot analysis showed that the level of p-p38 MAPK in the PNS female offspring rats was significantly increased (126.41 3.937, n = 6) compared to that in the control female offspring rats (101.35 3.468, n = 6, F = 12.162, P < 0.01). There was no significant difference in the level of p-p38 MAPK between the male control and PNS offspring rats (100.87 2.227, 104.25 4.364, respectively) (Fig. 2A and B).
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Fig. 2. (A) Effects of PNS on expression of p-p38 MAPK in offspring hippocampus. Representative immunoblot of p-p38 MAPK (top lane). Cytosolic fractions (50 mg protein/lane) from CON male (lane 1), CON female (lane 2), PNS male (lane 3) and PNS female offspring hippocampus (lane 4) were subjected to 10% SDS–PAGE and Western blot analysis using an anti-p-p38 MAPK antibody. Representative immunoblot for b-actin (low lane). (B) Summary of results of p-p38 MAPK in the hippocampus. Quantitative data based on the measurement of integrated optical density in Western blot illustrated in (A). The levels of p-p38 MAPK protein were significantly higher in PNS female offspring hippocampus than that of control female offspring hippocampus (P < 0.01).
3.2. Effects of PNS on p38 MAPK-positive expression in sub-regions of offspring hippocampus Immunohistochemistry demonstrated p38 MAPK immunoreactive cells in all principal neuronal populations of the hippocampus, namely pyramidal neurons in hippocampus subfields 1–4 (CA1–4), granule cells in the dense cell layer of the dentate gyrus (DG). Fig. 3A and B shows representative photomicrographs of total p38 MAPK staining in CON and PNS offspring hippocampus. The data was analyzed by selected the area of interest in pyramidal cell layer in CA1–4 and granule cell layer in dentate gyrus. Within CA3 and CA4 region, optical density of p38 MAPK was significantly increased above female control (51.901 3.081, 42.898 2.602, n = 6, respectively) in PNS female offspring hippocampus (65.836 2.49, 69.707 3.165, F 1 = 32.525, P1 < 0.001; F 2 = 38.458, P2 < 0.001, n = 6, respectively). Optical density of p38 MAPK was not significantly different between male control and PNS groups in CA1, CA3, CA4 fields and DG in offspring hippocampus (Fig. 4). There was no significant interaction between group and gender.
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Fig. 3. Immunohistochemical staining of offspring hippocampus using p38 MAPK antibody. A from female CON, B from female PNS. Or, oriens layer; Py, pyramidal layer; rad, radiatum layer; g, granular layer.
4. Discussion The mechanisms by which prenatal stress can program neuronal development with long-term consequences are not well understood. Within the developing brain, the limbic system (primarily the hippocampus) is particularly sensitive to
Fig. 4. Changes in p38 MAPK levels of offspring hippocampus by PNS treatments. The graph summarizes the results from immunostained images, n = 6, respectively. The effects of prenatal stress on the optical density of p38 MAPK-positive expression in each hippocampal sub-region and DG of female offspring. Immunodensity of p38 MAPK was significantly increased above female control in PNS female offspring hippocampal CA3 and CA4 fields (P < 0.001 vs. CON).
endogenous and exogenous corticosterone during development. The corticosterone level of offspring and dam caused by prenatal stress was positively correlated (Takahashi et al., 1998). The effects of PNS on expression of p38 MAPK in offspring hippocampal neurons were investigated in the study. The present results clearly show that the prenatal restraint stress induces significant increase in the expression of p-p38 MAPK and total p38 MAPK in female offspring hippocampus. The p38 MAPK are a family of Ser/Thr kinases that regulate important cellular processes such as stress responses, differentiation, and cell-cycle control. The p38 MAPK is activated via phosphorylation in neurones by a variety of stimuli including oxidative stress, excitotoxicity, and inflammatory cytokines (Johnson and Lapadat, 2002). Activated p38 MAPK can in turn induce phosphorylation of cytoskeletal proteins and activation of cytokines and nitric oxide, thus contributing to neurodegeneration. Over-activation of these stress-activated kinases is known to cause neuronal degeneration and impairment of the function of the central nervous system (Schroeter et al., 2003). PNS females had a higher overall adrenocorticotropic hormone response to restraint stress than do female controls, whereas PNS males showed faster recovery of stress-induced elevations in adrenocorticotropic hormone than did non-stressed males (McCormick et al., 1995). It was reported that there is a phase shift toward peak secretion of corticosterone in the light cycle in both sexes after PNS, but that only female had significant elevations of corticosterone during all phases of the light/dark cycle (Koehl et al., 1999). One of the mechanisms that could explain the differential effects between prenatally stressed males and females could be the gender difference in placental transport of GCs from the mother to the female fetus. Montano et al. (1993) found higher baseline serum corticosterone concentrations in female fetuses than in male fetuses which were due to a greater transport of corticosterone from maternal blood across the placenta of females and a greater binding of corticosterone in the placenta of male fetuses. Recently, Pivina et al. (2007) found prenatal social stress increased basal level of corticosterone in prenatal stressed female rats who have high stress reactivity of the hypothalamic–pituitary–adrenal axis, as well as a more profound effect on anxiety level and oestrous cycle. These reports indicate that effects of PNS on the HPA axis may be more predominant in female than in male offspring. Over-expression of p-p38 MAPK and total p38 MAPK in female offspring hippocampus may be related to high levels of expression for the N-methy-D-aspartate (NMDA) receptor and Ca2+ channel of female offspring hippocampus caused by PNS (Cai et al., 2007a,b). PNS rats have high levels of corticosterone. Corticosterone can prevent the intake of glutamate (Moghaddam et al., 1994) and increase the activity of N-methy-D-aspartate (NMDA) receptor (McEwen, 2000). It has been reported that hippocampal NMDA receptor expression was increased in hippocampus in adult rats following prenatal stress (Berger et al., 2002). It has been shown that glutamate induces apoptosis in hippocampal slices and it causes an impairment of cell viability that was dependent of ionotropic and metabotropic receptors activation and, may involve the activation of p38 MAPK pathway
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(Molz et al., 2008). It seems likely that the increase of the expression of p38 MAPK in offspring hippocampus by PNS increased extracellular glutamate and hippocampal NMDAR expression, which enhanced the intracellular Ca2+ and ROS (Zhu et al., 2004). ROS can increase the activity of p38 MAPK (Gupta et al., 1999). The mechanisms of prenatal restraint stress to cause increase of phosphorylation and total p38 MAPK in offspring hippocampus are not well known. For example, ‘‘Is corticosterone increase in prenatal offspring the limiting factor’’? ‘‘Does calcium influx through NMDA receptor result in ROS production’’? All of these questions are warranted for further study. In the study the expression of p38 MAPK increased significantly in CA3 of PNS offspring. Several lines of evidence could help to explain why the pyramidal neurons of CA3 are more vulnerable to PNS. First, granule cells of the dentate gyrus express a high density of both Type I and Type II corticosteroid receptors (Reul and de Kloet, 1985) and these neurons project heavily to CA3 pyramidal cells via mossy fiber output, which might make the neurons of CA3 more vulnerable to stress levels of corticosterone. Secondly, Somatic whole-cell current-clamp recording in CA3 pyramidal cells revealed 28 days of psychosocial stress exposure reduced the apparent membrane time constant and input resistance 20–25%, accompanied by increased amplitude of the hyperpolarization-induced voltage ‘‘sag.’’ All active membrane properties, including depolarization-induced action potential kinetics, complex spiking patterns, and after hyperpolarization voltages, were indistinguishable from control recordings (Kole et al., 2004). Previous quantitative morphological analysis of the dendritic architecture of Golgi-impregnated hippocampal neurons in prenatally stressed rats indicated that the study group had lower spatial learning capabilities along with decreased length and number of dendritic segments, branching of granules and CA3 pyramidal cells. There was no change in the dendritic morphology of CA1 pyramidal cells (Hosseini-Sharifabad and Hadinedoushan, 2007). Therefore, CA3 pyramidal neurons may be particularly vulnerable to PNS. In conclusion, the present result show that the prenatal restraint stress induces significant increase in the expression of p-p38 MAPK and total p38 MAPK in female offspring hippocampus. The data suggest that exposure of animals to a period of stressful experience during a critical phase could impose lasting effects on cellular signalling of offspring hippocampus. Acknowledgments We acknowledge the helpful comments and language editing of Dr. Edward Sharman at the University of California, Irvine, CA, USA. This work was supported by National Natural Science Foundation of China (No. 30670672). References Ahmed, T., Frey, J.U., Korz, V., 2006. Long-term effects of brief acute stress on cellular signaling and hippocampal LTP. J. Neurosci. 26, 3951–3958.
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Berger, M.A., Barros, V.G., Sarchi, M.I., Tarazi, F.I., Antonelli, M.C., 2002. Long-term effects of prenatal stress on dopamine and glutamate receptors in adult rat brain. Neurochem. Res. 27, 1525–1533. Bosch, O.J., Mu¨sch, W., Bredewold, R., Slattery, D.A., Neumann, I.D., 2007. Prenatal stress increases HPA axis activity and impairs maternal care in lactating female offspring: implications for postpartum mood disorder. Psychoneuroendocrinology 32, 267–278. Cai, Q., Zhu, Z.L., Huang, S.Y., Li, H., Fan, X.L., Jia, N., Zhang, B.L., Song, L., Li, Q.H., Liu, J.K., 2007a. Sex and region difference of the expression of ERK in prenatal stress offspring hippocampus. Int. J. Dev. Neurosci. 25, 207–213. Cai, Q., Zhu, Z.L., Li, H., Fan, X.L., Jia, N., Bai, Z.L., Song, L., Li, X., Liu, J.K., 2007b. Prenatal stress on the kinetic properties of Ca2+ and K+ channels in offspring hippocampal CA3 pyramidal neurons. Life Sci. 80, 681–689. Cannizzaro, C., Plescia, F., Gagliano, M., Cannizzaro, G., Mantia, G., La Barbera, M., Provenzano, G., Cannizzaro, E., 2008. Perinatal exposure to 5methoxytryptamine, behavioural-stress reactivity and functional response of 5-HT1A receptors in the adolescent rat. Behav. Brain Res. 186, 98–106. Gupta, A., Rosenberger, S.F., Bowden, G.T., 1999. Increased ROS levels contribute to elevated transcription factor and MAP kinase activities in malignantly progressed mouse keratinocyte cell lines. Carcinogenesis 20, 2063–2073. Hosseini-Sharifabad, M., Hadinedoushan, H., 2007. Prenatal stress induces learning deficits and is associated with a decrease in granules and CA3 cell dendritic tree size in rat hippocampus. Anat. Sci. Int. 82, 211–217. Johnson, G.L., Lapadat, R., 2002. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298, 1911–1912. Koehl, M., Darnaudery, M., Dulluc, J., Van Reeth, O., Moal, M.L., Maccari, S., 1999. Prenatal stress alters circadian activity of hypothalamo-pituitary– adrenal axis and hippocampal corticosteroid receptors in adult rats of both gender. J. Neurobiol. 40, 302–315. Kole, M.H., Cze´h, B., Fuchs, E., 2004. Homeostatic maintenance in excitability of tree shrew hippocampal CA3 pyramidal neurons after chronic stress. Hippocampus 14, 742–751. Lee, P.R., Brady, D.L., Shapiro, R.A., Dorsa, D.M., Koenig, J.I., 2007. Prenatal stress generates deficits in rat social behavior: reversal by oxytocin. Brain Res. 1156, 152–167. Maccari, S., Darnaudery, M., Morley-Fletcher, S., Zuena, A.R., Cinque, C., Van Reeth, O., 2003. Prenatal stress and long-term consequences: implications of glucocorticoid hormones. Neurosci. Biobehav. Rev. 27, 119–127. McCormick, C.M., Smythe, J.W., Sharma, S., Meaney, M.J., 1995. Sex-specific effects of prenatal stress on hypothalamic-–pituitary–adrenal responses to stress and brain glucocorticoid receptor density in adult rats. Brain Res. Dev. Brain Res. 84, 55–61. McEwen, B.S., 2000. The neurobiology of stress: from serendipity to clinical relevance. Brain Res. 886, 172–189. Moghaddam, B., Bolinao, M.L., Stein-Behrens, B., Sapolsky, R., 1994. Glucocorticoids mediate the stress-induced extracellular accumulation of glutamate. Brain Res. 655, 251–254. Molz, S., Decker, H., Dal-Cim, T., Cremonez, C., Cordova, F.M., Leal, R.B., Tasca, C.I., 2008. Glutamate-induced toxicity in hippocampal slices involves apoptotic features and p38 MAPK signaling. Neurochem. Res. 33, 27–36. Montano, M.M., Wang, M.H., Vomsaal, F.S., 1993. Sex-differences in plasmacorticosterone in mouse fetuses are mediated by differential placental transport from the mother and eliminated by maternal adrenalectomy or stress. J. Reprod. Fertil. 99, 283–290. Pivina, S.G., Shamolina, T.S., Akulova, V.K., Ordian, N.E., 2007. Sensitiveness to social stress in female rats with alteration of the pituitary-adrenal axis stress reactivity. Ross. Fiziol. Zh. Im. I. M. Sechenova 93, 1319– 1325. Reul, J.M., de Kloet, E.R., 1985. Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology 117, 2505–2511. Schroeter, H., Boyd, C.S., Ahmed, R., Spencer, J.P., Duncan, R.F., Rice-Evans, C., Cadenas, E., 2003. c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signaling in mitochondria-dependent apoptosis. Biochem. J. 372, 359–369.
540
Q. Cai et al. / Int. J. Devl Neuroscience 26 (2008) 535–540
Takahashi, L.K., Turner, J.G., Kalin, N.H., 1998. Prolonged stress-induced elevation in plasma corticosterone during pregnancy in the rat: implications for prenatal stress studies. Psychoneuroendocrinology 23, 571–581. Weinberg, J., Sliwowska, J.H., Lan, N., Hellemans, K.G., 2008. Prenatal alcohol exposure: foetal programming, the hypothalamic–pituitary–adre-
nal axis and sex differences in outcome. J. Neuroendocrinol. 20, 470– 488. Zhu, Z.L., Li, X., Chen, W.N., Zhao, Y., Li, H., Qing, C., Jia, N., Bai, Z.L., Liu, J.K., 2004. Prenatal stress causes gender-dependent neuronal loss and oxidative stress in the hippocampus of rats. J. Neurosci. Res. 78, 837–844.
The effects of prenatal stress on expression of p38 MAPK in offspring hippocampus Qing Cai a, Shuyun Huang a, Zhongliang Zhu b,c,*, Hui Li d, Qinghong Li d, Ning Jia c, Jankang Liu e,f a Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China College of Life Science, Northwest University, Xi’an, Shaan xi 710069, PR China c Department of Physiology and Pathophysiology, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaan xi 710061, PR China d Department of Pediatrics, Xi’an Jiaotong University First Hospital, Xi’an, PR China e Institute for Nutritional Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China f Institute for Brain Aging, University of California, Irvine, CA 92796, United States b
Received 27 April 2008; received in revised form 3 June 2008; accepted 3 June 2008
Abstract The hippocampus, which has the highest density of GC receptors in the brain, is involved in the regulation of the HPA and the behavioral responses to stress. Overexposure to corticosteroid hormones is harmful to hippocampal neuron integrity. Our purpose is to investigate the effects of prenatal stress (PNS) on expression of p38 mitogen-activated protein kinase (p38 MAPK) in offspring hippocampal neurons using Western blotting and Immunohistochemistry. The prenatal restraint stress induces significant increase in the expression of p-p38 MAPK and total p38 MAPK in female offspring hippocampus. The level of p-p38 MAPK in PNS female offspring rats was significantly increased (126.41 3.937, n = 6) compared with that in the control female offspring rats (101.35 3.468, n = 6, P < 0.01). Immunoblot analysis revealed there was significant difference in the level of total p38 MAPK between the female control and prenatal restraint stress offspring rats (101.70 3.162 vs. 128.111 2.724, respectively, P < 0.01). Immunodensity of p38 MAPK was significantly increased above female control in PNS female offspring hippocampal CA3 and CA4 fields (P < 0.001 vs. control group, CON). The data suggest that exposure of animals to a period of stressful experience during a critical phase could impose lasting effects on the offspring hippocampal neurons cellular signalling of offspring hippocampus. Crown Copyright # 2008 Published by Elsevier Ltd on behalf of ISDN. All rights reserved. Keywords: Prenatal stress; p38 MAPK; Hippocampus; CA3
1. Introduction Numerous studies show that stressors acting upon the organism during pregnancy can have distinct and long lasting effects on the offspring. Prenatally stressed offspring may exhibit alterations in brain morphology and behavior (Pivina et al., 2007; Lee et al., 2007), increased emotional reactivity (Bosch et al., 2007; Cannizzaro et al., 2008) and altered regulation of the hypothalamic–pituitary–adrenal (HPA) axis (Weinberg et al., 2008). It was reported hyperactivity of HPA axis in offspring induced by prenatal stress (PNS) was related to the high levels of maternal corticosterone secretion during
* Corresponding author at: School of Medicine, Xi’an Jiaotong University, Xi’an 710061, PR China. Tel.: +86 29 82655 669. E-mail address: [email protected] (Z. Zhu).
restraint stress (Maccari et al., 2003). The hippocampus, which has the highest density of glucocorticoid (GC) receptors in the brain, is involved in the regulation of the HPA and the behavioral responses to stress. A previous study reported that levels of phosphorylated mitogen-activated protein kinase and p38 MAPK, as well as phosphorylated calcium/calmodulindependent protein kinase II were enhanced shortly after swim stress (Ahmed et al., 2006). We have investigated the effects of PNS on the intracellular Ca2+ and reactive oxygen species (ROS) in hippocampal CA3 region of female offspring. PNS caused an increase in the concentrations of the intracellular calcium and the production of ROS in hippocampal CA3 region (Zhu et al., 2004). Our previous studies have demonstrated that PNS can increase the expression of p-ERK2 compared to that in the control female offspring rats and total ERK2 in female offspring hippocampus. ERK immunodensity was significantly increased in PNS groups
0736-5748/$34.00. Crown Copyright # 2008 Published by Elsevier Ltd on behalf of ISDN. All rights reserved. doi:10.1016/j.ijdevneu.2008.06.001
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Q. Cai et al. / Int. J. Devl Neuroscience 26 (2008) 535–540
in CA3 field in male offspring hippocampus compared with control animals. ERK optical density was significantly increased in PNS female offspring hippocampus CA1, CA3 and CA4 region (Cai et al., 2007a,b). Whether PNS causes the changes of p38 MAPK in offspring hippocampus neurons is unknown. In the present study Western blotting were employed to examine the effects of PNS on expression of p38 MAPK in offspring hippocampal neurons.
in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) for 1 h at room temperature and incubated with the primary antibody in TBSTwith 5% free-fat dry milk overnight at 4 8C with gentle shaking. The membranes were rinsed with TBST three times. The p38 MAPK levels were evaluated respectively with rabbit polyclonal antibodies that recognize unphosphorylated and phosphorylated p38 MAPK (1:1000 dilutions, Cell Signalling, Beverly, MA, USA) for 1 h at room temperature. Reactive proteins were detected with an ECL Western Blotting Detection Kit (AP Biotech, Buckinghamshire, UK). Protein loading was assessed by re-probing the blots with mouse anti-rat b-actin antibody.
2.3. Immunohistochemistry 2. Materials and methods 2.1. Animals and procedures All procedures were carried out in accordance with the United States Public Health Services Guide for care and use of laboratory animals and were approved by the Institutional Animals Care and Use Committee at Xi’an Jiaotong University. All efforts were made to minimize the number of animals used and their suffering. A total of 15 female and 5 male Sprague–Dawley rats were adopted. Female rats weighting 230–250 g and male rats weighting 280–350 g were used. Animals were provided by the experimental animal center of Xi’an Jiaotong University Medical College. The rats were housed in an animal room with controlled temperature (22 2 8C) and humidity (60%) controlled animal room on a 12 h light/dark cycle (light on from 8:00 a.m. to 8:00 p.m.) with free access to food and water throughout the experiment. Virgin female rats were placed overnight with adult male rats (3:1) for mating. Vaginal smear was examined on the following morning. The day on which the smear was spermpositive was determined as embryonic day 0. Each pregnant rat was then housed separately. A total of 12 pregnant rats were randomly assigned to prenatal stress (n = 6) and control groups (n = 6). The pregnant rats of PNS group were exposed to restraint stress on days 14–20 of pregnancy three times daily for 45 min (Koehl et al., 1999; McCormick et al., 1995). To prevent habituation of animals to the daily procedure, restraint periods were randomly shifted within certain time periods (08:00–11:00 a.m., 11:00 a.m.–02:00 p.m., and 04:00–07:00 p.m.). The restraint device was a transparent plastic tube (6.8 cm in diameter) with air holes for breathing and closed end. The length could be adjusted to accommodate the size of the animals. After birth, offspring of all groups were housed in the same animal room, and kept together with their biologic mothers. The pregnant rats of the control group were left undisturbed. On day 21, after all offspring were weaned, male and female pups were separated and housed four in each cage, respectively until testing at 1 month of age. At the end of postnatal day 30, one male and female offspring rat from the same dam have been selected with a random choice in two experiments. Six male and six female rats were used for Western blotting and Immunohistochemistry, respectively. The light phases in which data was collected.
2.2. Gel electrophoresis and Western blotting Six male and six female rats from different dams were decapitated under deep anesthesia with chloral hydrate (400 mg/kg, i.p.). The hippocampus was dissected out on the petri dish filled with ice and immediately frozen in liquid nitrogen and then kept in a 80 8C freezer until later experimental use. Each hippocampus was homogenized with a glass tissue grinder in 500 ml cold phosphate-buffered saline buffer containing protease inhibitors: 2 mg/mL aprotinine, 1 mg/mL leupeptine, and 1 mmol/L PMSF (St. Louis, MO, USA). The samples were centrifuged at 12,000 g for 20 min at 4 8C after homogenization. The total protein concentration in each sample was determined using the BCA protein assay reagent kit (Pierce, Rockford, IL, USA). The sample proteins (50 mg per lane) were separated by electrophoresis performed on 10% sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE, Bio-Rad, Barcelona, Spain) at room temperature. Following electrophoresis, the proteins in the gel were transferred to 0.45 mm polyvinyldifluoride membranes (PVDF). The membrane was blocked with 5% free-fat dry milk
Under deep anesthesia with chloral hydrate, the rats of each group were perfused transcardially with approximately 200 mL 0.1 mol/L PBS (pH 7.4), followed by approximately cold 400 mL 4% paraformaldehyde in PB (pH 7.4). The brains were removed, cut into blocks containing the hippocampus and post-fixed in the same solution for 24 h at 4 8C. Subsequently, the tissue blocks were dehydrated, processed for paraffin embedding and then cut into frontal sections 4 mm thickness. Brain sections were deparaffinized in xylene and hydrated through graded alcohols to Tris-buffered saline, washed with TBS three times (5 min each time). They were then treated with freshly made 3% H2O2 in TBS for 30 min. After washed several times in TBS, they were then treated with 0.1% Triton X-100. Nonspecific binding sites were blocked by incubation with normal goat serum 1 h at 37 8C. The sections were then incubated with polyclonal IgG anti-p38 MAPK (1:200 dilutions, Santa Cruz Biotechnology, CA) at 4 8C overnight. Biotinylated goat antirabbit IgG (1:500 dilutions, Beijing ZhongShan Biotechnology, China) used as the secondary antibody was incubated with the sections for 1 h at room temperature and detected with the streptavidin–peroxidase complex. The immunoreaction was visualized by treating the sections with DAB kit at room temperature. The sections were then dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped. Immunohistochemical controls were performed using the same procedures as described above except that the primary antibodies were omitted. No positive immunostaining was found in any controls (not shown). For qualitative evaluations of p38 MAPK immunoreactivity, the number of p38 MAPK and immunoreactive cells was counted using a computer-based imaging system (AnaliSYS, Soft Imaging System GmbH, Munster, Germany) with the images captured using a digital camera. The optical measurements were made blindly to the animal groups. Optical density (OD) was measured for estimation of quantitative differences in p38 MAPK immunoreactivity between experimental groups. OD analysis was performed on high resolution and analyzed with Scion Image Software based on NIH image (NIH Image, Bethesda, MD, USA). Background values were obtained from the neighboring white matter. The average of values from three sections for each subject was used for statistical analysis.
2.4. Statistics All values reported are mean S.E.M. Data analyses were performed using the software of SPSS 11.0. Two-way (prenatal treatment-gender) analysis of variance (ANOVA) was used to examine differences between male and female offspring and among the groups, and the interaction between group and gender. A difference was considered significant at P < 0.05 level.
3. Results 3.1. Effects of PNS on expression of phosphorylated p38 MAPK and total p38 MAPK in offspring hippocampus Phosphorylated p38 MAPK activation was analyzed using specific antibodies, by Western blotting from total hippocampal extracts. The results of Western blotting were shown in Fig. 1.
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Fig. 1. (A) Effects of PNS on expression of total p38 MAPK in offspring hippocampus. Representative immunoblot of p38 MAPK (top lane). Cytosolic fractions (50 mg protein/lane) from CON male (lane 1), CON female (lane 2), PNS male (lane 3) and PNS female offspring hippocampus (lane 4) were subjected to 10% SDS–PAGE and Western blot analysis using an anti p38 MAPK antibody. Representative immunoblot for b-actin to control protein loading (low lane). (B). Summary of results of total p38 MAPK levels in the hippocampus. Quantitative data based on the measurement of integrated optical density in Western blot illustrated in (A). The levels of total p38 MAPK protein were significantly higher in PNS female offspring hippocampus than that of control female offspring hippocampus (P < 0.01).
Each value was expressed relative to the mean density of the control group male rat. Mean density of protein bands in the control group was expressed as 100%. There were no differences of mean density of protein between the control group male and female rats. Immunoblot analysis of total p38 MAPK revealed that there was no significant difference in the level of p38 MAPK between the male control and PNS male offspring rats and control female offspring rats (100.57 3.409, 102.08 4.450, n = 6 for each group). Immunoblot analysis revealed there was significant difference in the level of total p38 MAPK between the female control and PNS offspring rats (101.70 3.162, 128.111 2.724, respectively, F = 12.547, P < 0.01) (Fig. 1A and B). Quantitative Western immunoblot analysis showed that the level of p-p38 MAPK in the PNS female offspring rats was significantly increased (126.41 3.937, n = 6) compared to that in the control female offspring rats (101.35 3.468, n = 6, F = 12.162, P < 0.01). There was no significant difference in the level of p-p38 MAPK between the male control and PNS offspring rats (100.87 2.227, 104.25 4.364, respectively) (Fig. 2A and B).
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Fig. 2. (A) Effects of PNS on expression of p-p38 MAPK in offspring hippocampus. Representative immunoblot of p-p38 MAPK (top lane). Cytosolic fractions (50 mg protein/lane) from CON male (lane 1), CON female (lane 2), PNS male (lane 3) and PNS female offspring hippocampus (lane 4) were subjected to 10% SDS–PAGE and Western blot analysis using an anti-p-p38 MAPK antibody. Representative immunoblot for b-actin (low lane). (B) Summary of results of p-p38 MAPK in the hippocampus. Quantitative data based on the measurement of integrated optical density in Western blot illustrated in (A). The levels of p-p38 MAPK protein were significantly higher in PNS female offspring hippocampus than that of control female offspring hippocampus (P < 0.01).
3.2. Effects of PNS on p38 MAPK-positive expression in sub-regions of offspring hippocampus Immunohistochemistry demonstrated p38 MAPK immunoreactive cells in all principal neuronal populations of the hippocampus, namely pyramidal neurons in hippocampus subfields 1–4 (CA1–4), granule cells in the dense cell layer of the dentate gyrus (DG). Fig. 3A and B shows representative photomicrographs of total p38 MAPK staining in CON and PNS offspring hippocampus. The data was analyzed by selected the area of interest in pyramidal cell layer in CA1–4 and granule cell layer in dentate gyrus. Within CA3 and CA4 region, optical density of p38 MAPK was significantly increased above female control (51.901 3.081, 42.898 2.602, n = 6, respectively) in PNS female offspring hippocampus (65.836 2.49, 69.707 3.165, F 1 = 32.525, P1 < 0.001; F 2 = 38.458, P2 < 0.001, n = 6, respectively). Optical density of p38 MAPK was not significantly different between male control and PNS groups in CA1, CA3, CA4 fields and DG in offspring hippocampus (Fig. 4). There was no significant interaction between group and gender.
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Fig. 3. Immunohistochemical staining of offspring hippocampus using p38 MAPK antibody. A from female CON, B from female PNS. Or, oriens layer; Py, pyramidal layer; rad, radiatum layer; g, granular layer.
4. Discussion The mechanisms by which prenatal stress can program neuronal development with long-term consequences are not well understood. Within the developing brain, the limbic system (primarily the hippocampus) is particularly sensitive to
Fig. 4. Changes in p38 MAPK levels of offspring hippocampus by PNS treatments. The graph summarizes the results from immunostained images, n = 6, respectively. The effects of prenatal stress on the optical density of p38 MAPK-positive expression in each hippocampal sub-region and DG of female offspring. Immunodensity of p38 MAPK was significantly increased above female control in PNS female offspring hippocampal CA3 and CA4 fields (P < 0.001 vs. CON).
endogenous and exogenous corticosterone during development. The corticosterone level of offspring and dam caused by prenatal stress was positively correlated (Takahashi et al., 1998). The effects of PNS on expression of p38 MAPK in offspring hippocampal neurons were investigated in the study. The present results clearly show that the prenatal restraint stress induces significant increase in the expression of p-p38 MAPK and total p38 MAPK in female offspring hippocampus. The p38 MAPK are a family of Ser/Thr kinases that regulate important cellular processes such as stress responses, differentiation, and cell-cycle control. The p38 MAPK is activated via phosphorylation in neurones by a variety of stimuli including oxidative stress, excitotoxicity, and inflammatory cytokines (Johnson and Lapadat, 2002). Activated p38 MAPK can in turn induce phosphorylation of cytoskeletal proteins and activation of cytokines and nitric oxide, thus contributing to neurodegeneration. Over-activation of these stress-activated kinases is known to cause neuronal degeneration and impairment of the function of the central nervous system (Schroeter et al., 2003). PNS females had a higher overall adrenocorticotropic hormone response to restraint stress than do female controls, whereas PNS males showed faster recovery of stress-induced elevations in adrenocorticotropic hormone than did non-stressed males (McCormick et al., 1995). It was reported that there is a phase shift toward peak secretion of corticosterone in the light cycle in both sexes after PNS, but that only female had significant elevations of corticosterone during all phases of the light/dark cycle (Koehl et al., 1999). One of the mechanisms that could explain the differential effects between prenatally stressed males and females could be the gender difference in placental transport of GCs from the mother to the female fetus. Montano et al. (1993) found higher baseline serum corticosterone concentrations in female fetuses than in male fetuses which were due to a greater transport of corticosterone from maternal blood across the placenta of females and a greater binding of corticosterone in the placenta of male fetuses. Recently, Pivina et al. (2007) found prenatal social stress increased basal level of corticosterone in prenatal stressed female rats who have high stress reactivity of the hypothalamic–pituitary–adrenal axis, as well as a more profound effect on anxiety level and oestrous cycle. These reports indicate that effects of PNS on the HPA axis may be more predominant in female than in male offspring. Over-expression of p-p38 MAPK and total p38 MAPK in female offspring hippocampus may be related to high levels of expression for the N-methy-D-aspartate (NMDA) receptor and Ca2+ channel of female offspring hippocampus caused by PNS (Cai et al., 2007a,b). PNS rats have high levels of corticosterone. Corticosterone can prevent the intake of glutamate (Moghaddam et al., 1994) and increase the activity of N-methy-D-aspartate (NMDA) receptor (McEwen, 2000). It has been reported that hippocampal NMDA receptor expression was increased in hippocampus in adult rats following prenatal stress (Berger et al., 2002). It has been shown that glutamate induces apoptosis in hippocampal slices and it causes an impairment of cell viability that was dependent of ionotropic and metabotropic receptors activation and, may involve the activation of p38 MAPK pathway
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(Molz et al., 2008). It seems likely that the increase of the expression of p38 MAPK in offspring hippocampus by PNS increased extracellular glutamate and hippocampal NMDAR expression, which enhanced the intracellular Ca2+ and ROS (Zhu et al., 2004). ROS can increase the activity of p38 MAPK (Gupta et al., 1999). The mechanisms of prenatal restraint stress to cause increase of phosphorylation and total p38 MAPK in offspring hippocampus are not well known. For example, ‘‘Is corticosterone increase in prenatal offspring the limiting factor’’? ‘‘Does calcium influx through NMDA receptor result in ROS production’’? All of these questions are warranted for further study. In the study the expression of p38 MAPK increased significantly in CA3 of PNS offspring. Several lines of evidence could help to explain why the pyramidal neurons of CA3 are more vulnerable to PNS. First, granule cells of the dentate gyrus express a high density of both Type I and Type II corticosteroid receptors (Reul and de Kloet, 1985) and these neurons project heavily to CA3 pyramidal cells via mossy fiber output, which might make the neurons of CA3 more vulnerable to stress levels of corticosterone. Secondly, Somatic whole-cell current-clamp recording in CA3 pyramidal cells revealed 28 days of psychosocial stress exposure reduced the apparent membrane time constant and input resistance 20–25%, accompanied by increased amplitude of the hyperpolarization-induced voltage ‘‘sag.’’ All active membrane properties, including depolarization-induced action potential kinetics, complex spiking patterns, and after hyperpolarization voltages, were indistinguishable from control recordings (Kole et al., 2004). Previous quantitative morphological analysis of the dendritic architecture of Golgi-impregnated hippocampal neurons in prenatally stressed rats indicated that the study group had lower spatial learning capabilities along with decreased length and number of dendritic segments, branching of granules and CA3 pyramidal cells. There was no change in the dendritic morphology of CA1 pyramidal cells (Hosseini-Sharifabad and Hadinedoushan, 2007). Therefore, CA3 pyramidal neurons may be particularly vulnerable to PNS. In conclusion, the present result show that the prenatal restraint stress induces significant increase in the expression of p-p38 MAPK and total p38 MAPK in female offspring hippocampus. The data suggest that exposure of animals to a period of stressful experience during a critical phase could impose lasting effects on cellular signalling of offspring hippocampus. Acknowledgments We acknowledge the helpful comments and language editing of Dr. Edward Sharman at the University of California, Irvine, CA, USA. This work was supported by National Natural Science Foundation of China (No. 30670672). References Ahmed, T., Frey, J.U., Korz, V., 2006. Long-term effects of brief acute stress on cellular signaling and hippocampal LTP. J. Neurosci. 26, 3951–3958.
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Berger, M.A., Barros, V.G., Sarchi, M.I., Tarazi, F.I., Antonelli, M.C., 2002. Long-term effects of prenatal stress on dopamine and glutamate receptors in adult rat brain. Neurochem. Res. 27, 1525–1533. Bosch, O.J., Mu¨sch, W., Bredewold, R., Slattery, D.A., Neumann, I.D., 2007. Prenatal stress increases HPA axis activity and impairs maternal care in lactating female offspring: implications for postpartum mood disorder. Psychoneuroendocrinology 32, 267–278. Cai, Q., Zhu, Z.L., Huang, S.Y., Li, H., Fan, X.L., Jia, N., Zhang, B.L., Song, L., Li, Q.H., Liu, J.K., 2007a. Sex and region difference of the expression of ERK in prenatal stress offspring hippocampus. Int. J. Dev. Neurosci. 25, 207–213. Cai, Q., Zhu, Z.L., Li, H., Fan, X.L., Jia, N., Bai, Z.L., Song, L., Li, X., Liu, J.K., 2007b. Prenatal stress on the kinetic properties of Ca2+ and K+ channels in offspring hippocampal CA3 pyramidal neurons. Life Sci. 80, 681–689. Cannizzaro, C., Plescia, F., Gagliano, M., Cannizzaro, G., Mantia, G., La Barbera, M., Provenzano, G., Cannizzaro, E., 2008. Perinatal exposure to 5methoxytryptamine, behavioural-stress reactivity and functional response of 5-HT1A receptors in the adolescent rat. Behav. Brain Res. 186, 98–106. Gupta, A., Rosenberger, S.F., Bowden, G.T., 1999. Increased ROS levels contribute to elevated transcription factor and MAP kinase activities in malignantly progressed mouse keratinocyte cell lines. Carcinogenesis 20, 2063–2073. Hosseini-Sharifabad, M., Hadinedoushan, H., 2007. Prenatal stress induces learning deficits and is associated with a decrease in granules and CA3 cell dendritic tree size in rat hippocampus. Anat. Sci. Int. 82, 211–217. Johnson, G.L., Lapadat, R., 2002. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298, 1911–1912. Koehl, M., Darnaudery, M., Dulluc, J., Van Reeth, O., Moal, M.L., Maccari, S., 1999. Prenatal stress alters circadian activity of hypothalamo-pituitary– adrenal axis and hippocampal corticosteroid receptors in adult rats of both gender. J. Neurobiol. 40, 302–315. Kole, M.H., Cze´h, B., Fuchs, E., 2004. Homeostatic maintenance in excitability of tree shrew hippocampal CA3 pyramidal neurons after chronic stress. Hippocampus 14, 742–751. Lee, P.R., Brady, D.L., Shapiro, R.A., Dorsa, D.M., Koenig, J.I., 2007. Prenatal stress generates deficits in rat social behavior: reversal by oxytocin. Brain Res. 1156, 152–167. Maccari, S., Darnaudery, M., Morley-Fletcher, S., Zuena, A.R., Cinque, C., Van Reeth, O., 2003. Prenatal stress and long-term consequences: implications of glucocorticoid hormones. Neurosci. Biobehav. Rev. 27, 119–127. McCormick, C.M., Smythe, J.W., Sharma, S., Meaney, M.J., 1995. Sex-specific effects of prenatal stress on hypothalamic-–pituitary–adrenal responses to stress and brain glucocorticoid receptor density in adult rats. Brain Res. Dev. Brain Res. 84, 55–61. McEwen, B.S., 2000. The neurobiology of stress: from serendipity to clinical relevance. Brain Res. 886, 172–189. Moghaddam, B., Bolinao, M.L., Stein-Behrens, B., Sapolsky, R., 1994. Glucocorticoids mediate the stress-induced extracellular accumulation of glutamate. Brain Res. 655, 251–254. Molz, S., Decker, H., Dal-Cim, T., Cremonez, C., Cordova, F.M., Leal, R.B., Tasca, C.I., 2008. Glutamate-induced toxicity in hippocampal slices involves apoptotic features and p38 MAPK signaling. Neurochem. Res. 33, 27–36. Montano, M.M., Wang, M.H., Vomsaal, F.S., 1993. Sex-differences in plasmacorticosterone in mouse fetuses are mediated by differential placental transport from the mother and eliminated by maternal adrenalectomy or stress. J. Reprod. Fertil. 99, 283–290. Pivina, S.G., Shamolina, T.S., Akulova, V.K., Ordian, N.E., 2007. Sensitiveness to social stress in female rats with alteration of the pituitary-adrenal axis stress reactivity. Ross. Fiziol. Zh. Im. I. M. Sechenova 93, 1319– 1325. Reul, J.M., de Kloet, E.R., 1985. Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology 117, 2505–2511. Schroeter, H., Boyd, C.S., Ahmed, R., Spencer, J.P., Duncan, R.F., Rice-Evans, C., Cadenas, E., 2003. c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signaling in mitochondria-dependent apoptosis. Biochem. J. 372, 359–369.
540
Q. Cai et al. / Int. J. Devl Neuroscience 26 (2008) 535–540
Takahashi, L.K., Turner, J.G., Kalin, N.H., 1998. Prolonged stress-induced elevation in plasma corticosterone during pregnancy in the rat: implications for prenatal stress studies. Psychoneuroendocrinology 23, 571–581. Weinberg, J., Sliwowska, J.H., Lan, N., Hellemans, K.G., 2008. Prenatal alcohol exposure: foetal programming, the hypothalamic–pituitary–adre-
nal axis and sex differences in outcome. J. Neuroendocrinol. 20, 470– 488. Zhu, Z.L., Li, X., Chen, W.N., Zhao, Y., Li, H., Qing, C., Jia, N., Bai, Z.L., Liu, J.K., 2004. Prenatal stress causes gender-dependent neuronal loss and oxidative stress in the hippocampus of rats. J. Neurosci. Res. 78, 837–844.