- Email: [email protected]
Effects of maternal stress during pregnancy on learning and memory via hippocampal BDNF, Arc (Arg3.1) expression in offspring
Accepted Manuscript Title: Effects of maternal stress during pregnancy on learning and memory via hippocampal BDNF, Arc (Arg3.1) expression in offspring Author: Su-zhen Guan Li Ning Ning Tao Yu-long Lian Ji-wen Liu Tzi Bun Ng PII: DOI: Reference:
S1382-6689(16)30089-8 http://dx.doi.org/doi:10.1016/j.etap.2016.04.012 ENVTOX 2498
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
9-11-2015 20-4-2016 23-4-2016
Please cite this article as: Guan, Su-zhen, Ning, Li, Tao, Ning, Lian, Yu-long, Liu, Ji-wen, Ng, Tzi Bun, Effects of maternal stress during pregnancy on learning and memory via hippocampal BDNF, Arc (Arg3.1) expression in offspring.Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2016.04.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of maternal stress during pregnancy on learning and memory via hippocampal BDNF, Arc (Arg3.1) expression in offspring
Su-zhen Guan1, Li Ning2, Ning Tao2, Yu-long Lian3, Ji-wen Liu2,*, Tzi Bun Ng4,*
1
Department of Social Medicine, College of Public Health, Xinjiang Medical
University, Urumqi 830011, China; 2
Department of Occupational Health and Environmental Health, College of Public
Health, Xinjiang Medical University, Urumqi 830011, China; 3
Department of Occupational Health and Environmental Health, College of Public
Health, College of Medical, Nantong University, Jiangsu 226000, China; 4
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of
Hong Kong, Hong Kong.
*
Corresponding authors:
Ji-wen Liu Tel no.: 86-9914365004 Fax no.: 86-9914365004 Email: [email protected] Tzi Bun Ng Tel no.: 852-39436872 Fax no.: 852-26035123 Email: [email protected]
1
Graphical abstract
Research Highlights Chronic stress in pregnancy affected spatial learning and memory of the offspring Plasma corticosterone level was increased The expression of Arc protein and BDNF protein and mRNA in offspring was attenuated
2
ABSTRACT The intrauterine environment has a significant long-term impact on individual’s life, this study was designed to investigate the effect of stress during pregnancy on offspring’s learning and memory abilities and analyze its mechanisms from the expression of BDNF and Arc in the hippocampus of the offspring. A rat model of maternal chronic stress during pregnancy was mating from 3rd day during been subjecting to chronic unpredictable mild stress (CUMS). The body weights and behavioral changes were recorded, and plasma corticosterone levels were determined by radioimmunoassay. The learning and memory abilities of the offspring were measured by Morris water maze testing from PND 42. The expression of hippocampal BDNF and Arc mRNA and protein were respectively measured using RT-PCR and Western blotting. Results indicated that an elevation was observed in the plasma corticosterone level of rat model of maternal chronic stress during pregnancy, a reduction in the crossing and rearing movement times and the preference for sucrose. The body weight of maternal stress’s offspring was lower than the control group, and the plasma corticosterone level was increased. Chronic stress during pregnancy had a significant impact on the spatial learning and memory of the offspring. The expression of BDNF mRNA and protein, Arc protein in offspring of maternal stress during pregnancy was attenuated and some relationships existed between these parameters. Collectively, these findings disclose that long-time maternal stress during pregnancy could destroy spatial learning and memory abilities of the offspring, the mechanism of which is related to been improving maternal plasma corticosterone and reduced hippocampal BDNF, Arc of offspring rats. Keywords: Maternal chronic stress; pregnancy; learning and memory; BDNF; Arc /Arg3.1; offspring
3
1. Introduction In the daily life, people often encounter different kinds of psychosocial stress, even at some specific physiological stages. It is difficult to avoid acute or chronic psychological stress, which may have a significant impact on health. Some women also experience daily stress during pregnancy, these stresses include depression, anxiety, anger, day-to-day challenges, sudden change of environment, social isolation, and so on. Maternal stress during pregnancy has been implicated as one of the risk factors for development in the offspring (Kramer et al., 2009; Le Moal, 2007). A large number of epidemiological evidence has indicated that a variety adverse pregnancy outcomes appear during maternal stress: the higher of the maternal plasma cortisol levels, birth rate of low birth weight babies and preterm birth (Gale and Martyn, 2004; Kajantie et al., 2005). It may also cause a rise in disruption of sleep patterns, reduction of deep sleep time, abnormality in sleep time, and prolongation of crying time (Leung et al., 2010). In addition, it can also have an effect on children's later growth and lead to weaker attentiveness in school-age children, emotional and behavioral problems and poor learning and memory abilities (Lobel et al., 2008). Recent studies have indicated that stress experiences of fatal stage affect synaptic plasticity and neurogenesis in specific areas of the central nervous system, particularly in the hippocampus (Aisa et al., 2009), which is part of the limbic system in brain and has a major role in cognition and mood regulation. Some studies have shown that stress during pregnancy inhibits cell proliferation in the hippocampus of offspring which causes impairment of learning and memory (Aguilera, 2011). The brain-derived neurotrophic factor (BDNF), which is directly involved in many physiological aspects of central nervous system such as neurite growth and neuronal survival (Kozisek et al., 2008), and plays a key role in learning and memory. Other data suggest that BDNF is also involved in both short- and long-term plasticity of glutamatergic synapses. BDNF signaling enhances synaptic maturation and increases synaptic density in the hippocampus, the synthesis and secretion of BDNF in the continue to be regulated by activity (Gronli et al., 2006). Another important neural basis of learning and memory is immediate early genes (IEGs), which is a kind of 4
encoding transcription factor protooncogene. A new kind of IEGs, activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), has recently gained a significant amount of attention. When the synaptic activity was increased, the expression of Arc/Arg3.1 in hippocampal neuron dendrites was significantly increased after stress, which was directly involved in synaptic plasticity and memory consolidation (Shepherd and Bear, 2011). The intrauterine environment has a significant long-term impact on individual’s life, it was hypothesized that maternal stress during pregnancy will have a negative effect on the offspring’s learning and memory, at the same time, exposure of the fetus in higher levels of corticosterone (It is a glucocorticoid in rodents and the primary end product of the hypothalamic– pituitary– adrenal axis) and the changes of expression of hippocampal BDNF and Arc maybe is the important reason for learning and memory. Therefore, this study was designed to investigate the effect of stress during pregnancy on
offspring’s learning and memory and analyze its mechanisms from
the expression of BDNF and Arc in the hippocampus.
2. Materials and methods 2.1. Animals Twenty female
Wistar rats weighing 240~270g and fifteen male Wistar rats
weighing 300~350g and sexually mature [all supplied by the Animal Laboratory Center of Xinjiang Medical University, Urumqi, Xinjiang, China; experimental animal certificate number: SCXK (new), 2011-000.], which were randomly allocated into seven cages (5 rats in each cage, male and female apart) after acclimatization for a week. All rats were maintained under standard laboratory conditions (12 h light/dark cycle, temperature 21-23℃, relative humidity 45-65%, and food and water ad libitum) during this week. 2.2. Chronic unpredictable mild stress (CUMS ) procedure The CUMS procedure followed a previously described method (Willner, 1997) with minor modifications. Chronic unpredictable mild stress comprised exposure to the following stressors in a random sequence everyday for 3 weeks: food deprivation 5
for 24 h; water deprivation for 24 h; cage tilt (45◦,7 h); noise housing (1500 Hz, 92 db, for 1 h); behavioral restriction for 4 h; forced swimming for 1 h in a 31℃ water bath; squeezing tail for 1 min; shaking stress ( 30 min); hot stress in an oven (42℃, 5 min); soiled cage (200 ml of water poured into the bedding, 8 h). One of the ten different stressors was randomly administered each day. During the process, the model rats were moved into another room (light intensity and temperature of two rooms were basically the same), then back to the room after the stimulation. 2.3. The treatments of maternal stress rat during pregnancy The maternal stress rat during pregnancy was mating from 3rd day during subjected to CUMS (as shown in the figure below). Twenty female Wistar rats were randomly allocated into two groups (10 rats per group), namely, maternal chronic stress rat during pregnancy model group (PS group) and control group (PC group). Before gestation, PS group was mated with a male in one cage, two rats in PC group were mated with a male rat in one cage. All female rats were examined every morning and pregnancy was confirmed by sperm positivity. Then male and female rats were separated after female conception, which was designated gestational day 0 (GD 0). After gestation, there were also 10 rats in each group. PC rats were housed with 5 in each cage (1 per cage after GD18), while PS rats were housed individually (1 per cage). Every stress factors didn’t suspend during mating. All rats were maintained under standard laboratory conditions.
Fig. Experimental procedure
6
2.4. Measurement of CUMS model 2.4.1. Measurement of plasma corticosterone level Venous blood samples (1 ml) were collected from the rats on the day before stress (Baseline) and then on the 1st, 7th, and 14th. Plasma was used for determination of plasma corticosterone level. Blood samples were centrifuged (3000 rpm for 20 min at 4℃), and the plasma obtained was stored at −35℃. Corticosterone was measured using a radioimmunoassay (RIA) kit in accordance with the manufacturer’s instructions (Coat-A-Count, Diagnostics Products Corporation). The intra-assay variability of the RIA ranged between 3.2% and 4.7%. The plasma corticosterone levels were derived from the determined cortisol values using the following conversion formula: Corticosterone concentration=Cortisol concentration ×50 (Liu et al., 2004). 2.4.2. Open-field test An open-field method was used to conduct praxeological scoring in the two groups of rats. The open-field device was made of opaque materials with a 80 cm × 80 cm square, located at the bottom, which was divided equally into 25 equilateral squares. Surrounding it was a wall with a height of 40 cm. The rat was put in the central square, and the number of squares the rat traversed in 3 min was recorded (only the squares on which the rat landed with four legs could be numbered as the score of horizontal activity) and the duration of standing on hind limbs was noted. Each rat was measured once for 3 min. A score was given by each of the two observers and the average value was taken. The percentage time spent in this central zone is considered indicative of exploratory behavior and may reflect a decrease in anxiety, although this parameter is not sensitive to all anxiolytics and may not model certain features of anxiety disorders (Li et al., 2010). The results reflect mean values of daily tests over three days. 2.4.3. Sucrose preference test In the sucrose preference test, the animals were allowed to consume water and a 1% sucrose solution for 1 h after food and water deprivation for 20 h, following 48 h of exposure to both water and sucrose solution. The positions of the two bottles (right/left) were varied randomly across animals and were reversed after 30 min. 7
Sucrose preference was calculated according to the following ratio: sucrose preference (%) = [sucrose intake (g)/sucrose intake (g) + water intake(g)]×100% (Prut and Belzung, 2003). The results reflect mean values of daily tests over three days. 2.5. Allocation of the offspring into groups The day on which the offspring were born was designated as postnatal day 0 (PND 0). The offspring were weaned from their mothers on PND 21, and male and female offspring separated. They remained undisturbed and were allocated to two groups, consisting of 8 male vs. 8 female offspring with PS, and 8 vs. 8 with PC, respectively. The 16 offspring from PS were called PS offspring, and offspring from PC were called PC offspring. Offsprings were housed in cages with up to 8 rats. 2.6. Measurement of offspring’s learning and memory abilities--Morris water maze testing(MWM) 2.6.1. Testing room and apparatus The water maze apparatus consisted of a circular pool (1.54 m diameter) made of stainless steel. The pool was filled with water (25±1℃) that was made opaque by the addition of non-sugar milk powder. During MWM training, an escape platform (20 cm diameter) made of stainless steel with a grooved surface was submerged 5 cm under the water level. All tests were carried out in the same experimental room and the MWM testing area was isolated from the operator and computer by a white curtain. Dark boards of different shapes provided landmarks in the testing room. Three days prior to the commencement of the acquisition phase, offspring underwent training to acclimatize them to the testing room and pool. The platform was placed in the centre of the pool and offspring were placed on the platform three times for 15 s. 2.6.2. Acquisition phase and reversal task All animals were tested in MWM started from PND 42 for five consecutive days. On each day, animals received two sessions of testing, at 10:00～12:00 am and 18:00～20:00 pm. Each session consisted of four trials with an inter-trial period of 10 min. For each trial, the animal was released from 1 of the 4 cardinal compass points (N, S, E, W). The animal was allowed a maximum of 120 s to locate and mount the escape platform with a post-trial timeout of 15 s on the platform. Escape latency (EL), 8
the time of locating the platform was our record time. The last EL was the average of EL from four different quadrants respectively in 5 days. After the test, the platform was removed, the offspring were put into the water from the same entry to nod to the wall, allowed to swim for 2 min. Closed the window and the curtains, fixed facilities of the room, in order to make sure that there was not any clue to mark the position of platform. Need to determine an effective positioning capability of the fixed structure within and outside the maze as a clue. Another index, the number of crossing platform, refers to the number of offspring shuttling the platform through the original position after removing the platform in a certain period of time. 2.7. Tissue collection from offspring For all subjects, the brain was removed after determination of learning and memory ability. Following intraperitoneal injection of chloral hydrate anesthesia, the hippocampus was excised from the brain, placed on a freezing aluminum dissection stage, bisected midway between the septal and temporal poles. One half of the left hippocampus was fixed with 4% formalin solution for HE sliced pathology (the change of structure of hippocampal CA1 area), and half of them were fixed with glutaraldehyde for electron microscopy. Then rapidly put right hippocampus frozen in liquid nitrogen and dissected on ice-cold glass plates, frozen on dry ice and stored at -80℃. The principle of using 50% male and 50% female was followed during the process. 2.8. Quantitative real-time RT-PCR Total RNA was extracted from 50-100 mg hippocampus according to the instructions of “RNA-BeeTM isolation of RNA” kit (TRIzol® Reagent, Life Technologies, USA). Extracted RNA was quantified by Nucleic acid protein quantitative instrument. Samples with the A260/280 ratio between 1.8-2.0 were used. Briefly, after homogenization of the tissue in reaction mixture containing RNA-Bee and chloroform and centrifugation at 12 000×g for 15 min at 4℃, the extracted RNA
9
in the aqueous phase was obtained. 200 µl of this aqueous phase was mixed with the same volume of isopropanol and after centrifugation at 12 000×g for 15 min at 4℃, RNA was precipitated and formed a pellet. The pellet was washed with 75% ethanol (by vortexing and centrifugation at 7 500×g for 5 min at 4℃) and then ethanol was removed and the pellet of RNA was allowed to dry, and then solubilized in 25 µl DEPC water. RNA concentration and purity were evaluated by spectrometry by optical density (OD) measurements at 260 -280 nm. A two-step reverse-transcription quantitative PCR (RT-qPCR) assay was performed, and both reactions [reverse transcription (RT) and quantitative polymerase chain reaction (qPCR)] were performed in a Chromo 4 Real Time PCR Instrument (MJ Research, USA). cDNA was generated from 600 ng of total RNA in a total volume of 40 μl using a cDNA synthesis kit (ReverttAid First Strand cDNA Synthesis Kit, USA). PCR was performed using the SYBR® Select Master Mix reagent for performing realtime PCR assays for the genes encoding BDNF and Arc. RT-PCR primers used were as follows: BDNF (NM_001270630.1): forward, 5'- AGGCACT GGAACTCGCAATG-3', reverse, 5'-AAGGGCCCGAACATACGATT-3'; Arc (NM_019361.1): forward, 5'-CCCATCTATGAGGGTTACGC-3', reverse, 5'-TTTAATGTCACGCACGATTT C-3'; β-actin (NM_031144.3): forward, 5'- CCCATCTATGAGGG TTACGC-3', reverse, 5'- TTTAATGTCACGCACGA TTTC-3'. The thermal cycling conditions were as follows: 2 min at 50℃ and 10 min at 95℃ followed by 40 cycles of 95℃ for 15 s and 60℃ for 1 min. The threshold value (Ct) for each sample was set in the exponential phase of PCR, and the ΔΔCt method was used for data analysis. β-action was used as reference gene. The experiment was performed in triplicate. 2.9. Western blotting Following tissue homogenization, protein concentration was assayed using a bicinchoninic acid (BCA) test (Beijing Tiangen, China). Punches were sonicated in 120-150 μl ice-cold RIPA buffer containing 50 mM Tris-HCl (pH 8.8), 150 mM NaCl, 1% NP-40 and 0.1% SDS (Beijing Applygen Technologies Inc, Beijing, China). The homogenate was then centrifuged at 12 000 g for 5 min, and the supernatant saved for analysis. Protein concentrations were determined using the BCA assay (Pierce, 10
Rockford, IL). Sample buffer was immediately added to the homogenates, and the samples were boiled for 5 min. Protein extracts (30 μg) were then electrophoresed in 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Blots were blocked in TBS buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.05% Tween 20) with 5% dry milk and incubated with an anti-Arc/Arg3.1 antibody (1:100; Santa Cruz Biotechnology), anti-BDNF antibody (1:200; Santa Cruz Biotechnology). Blots were then incubated with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:10 000; Santa Cruz Biotechnology) for 1 h at room temperature and developed using the West Dura chemiluminescent substrate (Pierce Laboratories). Densitometry was determined based on band intensity, and relative protein expression was quantified by densitometry using the Total Lab 2.01 analysis system (Phoretix, UK). To control for inconsistencies in loading, optical densities were normalized to β-action protein expression. Data for treated animals were normalized to the average value of the naive controls. 2.10. Statistical analysis Statistical analysis of all data, expressed as mean ± SD, was performed using the Statistical Package for the Social Sciences 13.0, and all graphs were constructed in GraphPad Prism 5.0. Differences between the means of maternal data in plasma corticosterone level, open-field test, sucrose preference test and the performance of offspring in MWM were analyzed using repeated measurement analysis of variance, LSD-t test to make multiple comparison at the different time. All RT-PCR and Western blotting data were analysed using student’s t test for comparison between two offspring groups. Bivariate correlations were performed using Spearman correlations. P value less than 0.05 was considered to be statistically significant.
3. Results 3.1. CUMS elevated maternal plasma corticosterone level The repeated measurement analysis of variance showed that chronic stress had a significant impact on the circulatory corticosterone level (F=14.996, P=0.001). Corticosterone level of the PS group drastically changed with stress time (F=64.607, 11
P<0.001). At the same time, there was an interactive relationship between time and stress factors (F=8.144, P=0.001). LSD-t test revealed that plasma corticosterone level of the PS group rose to the peak value which was higher than that of the PC group following exposure to stress for 7 days (t=9.378, P<0.001). However, the plasma corticosterone level of the PS group declined after exposure to stress for 14 days, but still remained higher than that of the PC group (t=4.437, P<0.001), indicating that the PS group was in a stressful state. Plasma corticosterone levels did not reach statistical significance between rats in the PC group and the PS group at the time of the baseline (t=1.128, P=0.130) and after exposure to stress for 1 day (t=0.738, P=0.235) (Figure1).
3.2. CUMS reduced horizontal and vertical movements in PS rats The repeated measurement analysis of variance revealed that chronic stress significantly affected the horizontal and vertical movements in the PS group (F =12.347, 8.541; all P<0.001). After exposure to stress for 21 days, both horizontal movement (Figure 2A) and vertical movement (Figure 2B) in the PS group were lower than those of the PC group (t day21=3.828, 3.535; all P<0.001 ) by LSD-t test. However, no significant differences between the PS group and the PC group were observed at baseline and after exposure to stress for 1, 7, 14 day (t P=0.218, 0.310. t 0.132. t
day14
day1=0.313,
0.288; P=0.379, 0.388; t
day7
day0=0.796,
0.497;
=0.827, 1.565; P=0.417,
=1.651, 1.777; P=0.113, 0.089.) (Figure 2). The reduction of horizontal
movements reflects that animal's activity reduced, the vertical motion reflects less curiosity about the new environment in which the animals was subjected to.
3.3. CUMS decreased Liquid consumption in PS rats The repeated measurement analysis of variance revealed that chronic stress significantly affected sugar water consumption, total liquid consumption and 1% sucrose preference in the PS rats (F=11.819, 6.966, 10.548; all P<0.001). It showed that stress affected fluid consumption index of rats during pregnancy. Regarding the baseline liquid consumption index, there was only a slight difference between the PS 12
and PC rats (P>0.05). A comparison of each point in time revealed that after 1 day of stress, sugar water consumption underwent a significant decline among rats in the PS group and the PC group by LSD-t test (all P<0.05). Following exposure to stress for 7, 14 and 21 day, it was manifested that sucrose-intake of the PS rats reduced compared with PC rats by LSD-t test (all P<0.05). Similarly, pure water consumption of the model group was higher than the PC group by LSD-t test (all P<0.05) (Figure 3).
3.4. CUMS reduced the rate of increase of maternal body weight The repeated measurement analysis of variance revealed that chronic stress significantly affected the rate of increase of maternal body weight in the PS rats (F=4.854, P=0.044), indicating that stress had a negative effect on rat weight gain during pregnancy. Along with the prolongation of the stress time, the rate of weight increase in rats of the model group was lower than the control group (F=85.191, P<0.001). Following exposure to stress for 7, 14 and 21 day, the body weight of PC rats was higher than rats in the PS group by LSD-t test (all P<0.05). However, no significant differences between the PS group and the PC group were observed at baseline and after stress for 1 day (P >0.05) (Figure 4).
3.5. Reproductive ability was impaired in PS rats There was statistically significant difference between the PS group and the PC group in the number of offspring and days of pregnancy (t=3.145, P=0.012; t=2.186, P=0.042) using student’s t test. The number of offspring (Figure 5A) and days of pregnancy (Figure 5B) in PS group was less than those of the PC group, indicating that maternal stress during pregnancy rat adversely influenced the reproductive ability of them. No significant differences were observed in sex distribution between two groups (t=0.798, P=0.440) (Figure 5C) (Figure 5).
3.6. Body weight and plasma corticosterone level of offspring changed after maternal stress during pregnancy On PND 28 and 42, the weight of offspring in PS group were lower than PC 13
(t=4.391, P<0.001; t=1.966, P=0.001) using student’s t test, showing that maternal stress during pregnancy affected the growth rate of offspring as seen in the body weight (Figure 6A). Using student’s t test, the plasma corticosterone level in PS offspring was higher than that in PC offspring on PND 28 and 42 (t=-2.251, P=0.035; t=-3.767, P=0.001), indicating that plasma corticosterone level in offspring of maternal stress during pregnancy was elevated (Figure 6B) (Figure 6).
3.7. Spatial learning and memory of
offspring in MWM changed after maternal
stress during pregnancy The repeated measurement analysis of variance showed that chronic stress during pregnancy had a significant impact on the escape latency of offspring from PND 42 (F=7.578，P<0.001), the escape latency of PS offspring were higher than that of PC. The escape latency of the PS offspring drastically changed with stress time (F=64.682，P<0.001). At the same time, there was no interactive relationship between time and stress factors (F=1.444，P=0.228). LSD-t test revealed the escape latency of the PS offspring group was higher than that of the PC offspring following measured 1, 4 and 5 days (Figure 7A) (tday1=2.598, P=0.004; tday4=2.638, P=0.003; tday5=8.755, P<0.001). However, escape latency did not differ between offspring in the PC group and the PS group at the time of measuring 2 and 3 days (tday2=0.569, P=0.287; tday3=0.309, P=0.380). The number of crossing platform of PS offspring was lower than that of PC by independent sample t-test (t=2.919, P=0.003) (Figure 7B). The results suggested that maternal stress during pregnancy had a significant impact on the spatial learning and memory of
offspring from the escape latency and crossing
platform in MWM (Figure 7).
3.8. Hippocampal structure of offspring damaged due to Maternal stress during pregnancy Observed under HE pigmentation with light microscope, each layer of hippocampus structure in PS offspring was not clear, cell morphology was irregular, 14
the gap of cell increased and arranged loosely (Figure 8A), suggested that structure of hippocampal CA1 area of offspring in maternal stress during pregnancy been changed. The results of electron microscope showed that the hippocampal neurons of PS offspring changed such as nuclear membrane invagination, reduction of nucleolar volume, nuclear vacuolated structure (Figure 8B). A series of changes in hippocampal synaptic cell were found, including granules sparsity, degranulation , the quantity of ribosome been reduced, the presynaptic membrane particles decreased in PS offspring compared with PC control group (Figure 8C). 3.9. Maternal stress during pregnancy effects on Arc and BDNF expression in offspring’ hippocampus from RT-PCR and Western blotting Determination of the extracting RNA by ultraviolet spectrophotometry showed that the ratio of A 260/A 280 lied between 1.8 ~ 2.0 and two clear bands in 28 s and 18 s were revealed after formaldehyde agarose gel electrophoresis. Strip quantity of 28 s was double that of 18 s, suggesting that RNA samples were intact (Figure 9A). Compared with PC offspring, the average expression of BDNF mRNA in PS offspring was significantly decreased (t=3.794, P=0.004) as shown by RT-PCR (Figure 9B). However, no significant differences between the PS offspring and the PC offspring were observed at the average expression of Arc (t=1.624, P=0.153). The molecular weights of BDNF and Arc were respectively 14 kDa and 50 kDa according to Western blotting results (Figure 9C). The average optical density (OD) values were displayed: statistically significant differences in hippocampal BDNF and Arc protein between PS offspring and PC offspring were detected (t=28.107, P=0.001; t=6.030, P=0.026) (Figure 9D). Compared with the PC offspring, the hippocampal BDNF and Arc protein of PS offspring were reduced (Figure 9).
3.9. Correlations of learning and memory of offspring and the determination results of their mothers
15
When all parameters of the PS offspring and their mothers were included in the correlation analysis, the escape latency of PS offspring was found to be positively correlated with the plasma corticosterone level of their mothers (r=0.556, P<0.05), and negatively correlated with horizontal movement and sucrose preference of their mother (r=-0.464, -0.626; all P<0.05). Meanwhile, crossing platform of the PS offspring was found to be positively correlated with horizontal movement and sucrose preference of the mother (r=0.676, 0.630; all P<0.05) and negatively correlated with the plasma corticosterone level of the mother (r=-0.441, P<0.05) (Table 1 ). 3.10. Correlations of learning and memory and hippocampal protein expression of offspring The escape latency of PS offspring was found to be negatively correlated with BDNF and Arc protein expression (r=-0.589, -0.421; all P<0.05). Meanwhile, crossing platform of PS offspring was found to be positively correlated with BDNF and Arc protein expression (r=0.556, 0.458; all P<0.05) (Table 2 ).
4. Discussion It is well known that many factors during pregnancy can provoke lead behavioral dysfunctions and dramatic developmental retardations. In the late 1980s, the chronic unpredictable mild stress (CUMS) model, originally developed by Paul Willner and colleagues, and based on both clinical and preclinical research regarding the etiology of stress (Willner, 1997), has been shown to mimic daily hassles and stress levels in humans. We started mating experiment during 3rd day in 21 days model period. Our experimental data showed a higher plasma corticosterone (stress hormone) level of rats in the PS group than that of PC rats, indicated that the model of maternal stress during pregnancy was established successfully. We also found that rat activity, curiosity to a new environment and reward reaction after chronic exposure to stress for 21 days during pregnancy were all diminished through behavioral and liquid consumption testing. Above all, stress during pregnancy can induce changes in the maternal neurobiological variables (Hsu et al., 2012; Yang et al., 2013). At the same time, our study clearly demonstrates that stress has an effect on 16
maternal weight gain, resulting in the increased plasma corticosterone levels of mothers, that can curb weight growth, accelerate protein decomposition and suppress protein synthesis (Abdul Aziz et al., 2012). The fetal function of the HPA axis was reprogrammed when the period of fetal exposure to high GC conditions, highly consistent with a considerable amount of related experimental results (Kapoor et al., 2006), such as, the embryo resorption, deformity, fetal growth restriction, low birth weight, and even the changes of sex ratio (Griffin et al., 2003). In human studies, maternal cortisol is known to cross the placenta and likely influence fetal development (Foster et al., 2008; Obel et al., 2005). Therefore, we found that the number of offspring and days of pregnancy in PS group was less than the PC group. Excess cortisol alters the normal functioning of the HPA axis, which may last for a lifetime (Weinstock, 2005). However, high levels of corticosteroids after stress damage the hippocampus, as it contains an elevated concentration of adrenal cortical hormone receptor in brain (McLaughlin et al., 2007) and selectively plays a role in the hippocampus, which has a pivotal function in learning and memory of rodents (Li et al., 2014; Salomon et al., 2011) . At the same time, stress can cause hippocampal neurons to change, hippocampal dendrites of pyramidal neurons shrink, which could damage the memory (Kosten et al., 2007;Carvalho-Netto et al., 2011). Our findings support the initial hypothesis drawn hippocampal structure changed in PS offspring. In particular, the present study is to assess and compare the effect of maternal stress during pregnancy on MWM performance from the offspring’s age of 42 days. Results showed that offspring whose mothers had been exposed to series of mixed stressors during pregnancy demonstrated an increased latency to find the platform and a decreased number of crossing platform, signified that maternal stress during pregnancy had a significant adverse impact on the spatial learning and memory of offspring. Similar to the findings of a more recent study, prenatal stressed rat offspring demonstrated increased latencies to find the platform in the MWM which was associated with the use of a random search strategy (Amugongo and Hlusko, 2014; Rizk et al., 2006). These findings are in line with suggestive mechanisms that glucocorticoids had been shown to affect MWM performance (Yu et al., 2004), which 17
affected the hippocampus: acute high levels facilitate hippocampal neurogenesis, synaptogenesis and dendritic remodeling, but chronically elevated levels impair these processes (Khalil et al., 2005). In rats, maternal stress during gestation increased latency in the MWM and this was associated with decreased LTP in hippocampal CA1 region slices (Kapoor et al., 2006) To determine the mechanisms underlying the effects of maternal stress during pregnancy on cognition in offspring, we focused on two molecules involved in the memory processes—BDNF and Arc/Arg3.1 (related to learning and memory). BDNF has been shown to play diverse roles in modulating the structure and function of the brain (Kozisek et al., 2008;Gronli et al., 2006). BDNF regulates dendritic and axonal morphology and affects synaptogenesis and synaptic transmission. Studying revealed that the average expression of BDNF in PS offspring was significantly decreased. The HPA axis is overactive when subjected to higher glucocorticoids, leading to BDNF down regulation, damage of mitochondria through Glu-NMDAR-NOS pathways, leading to atrophy of hippocampal neurons, apoptosis, and reduction of dentate gyrus granular cell regeneration, etc (Sirianni et al., 2010). In turn, BDNF has been reported to be an attractive candidate that translates experience-dependent neuronal activity into structural and functional changes in neuronal populations under prenatal stressful conditions (Maioli et al., 2012). Induction of immediate early genes (IEGs) is viewed as an important step in the formation of long-lasting neuro-adaptations underlying learning and memory (Inberg et al., 2013; Moron et al., 2010). Among the effector IEGs, Arc/Arg3.1 is a unique IEG that may be induced by neuronal activity and specifically trafficked and localized to recently potentiated synapses, where it may interact with structural proteins and proteins critical to synaptic plasticity (Li et al., 2009). Our present results showed that Arc/Arg3.1 protein expression of hippocampus decreased, compared with PC offspring. Zhong et al. (Zhong et al., 2008) previously reported that old mice showed impaired induction of Arc and mild hippocampus- dependent memory deficits in the MWM test. The possible reason was that higher glucocorticoids of offspring after maternal stress during pregnancy has a regulatory effect on Arc/Arg3.1 expression. 18
Moreover, chronic stress should inhibit Arc protein expression in the hippocampus, which mediates expression of AMPA receptor, the hippocampus cell swallow phenomenon appears, then can damage its own stable synaptic plasticity and impair hippocampal function. Thirdly, It is known that Arc expression is stimulated by learning-induced neuronal activity, N-methyl-D-aspartic acid receptor complexes (NMDARs) activation and LTP as well as by BDNF-TrkB signaling (Maioli et al., 2012). Apart from the HPA axis, signaling through NMDARs is critically involved in learning, memory and synaptic plasticity (Cho et al., 2009). Combined with our finding that the learning and memory of PS offspring correlated with BDNF and Arc protein expression, these findings raise the possibility that BDNF and Arc can promote synapse-specific translation during LTP production or memory formation (Fumagalli et al., 2009). As expected, the present study demonstrated that maternal stress during pregnancy could destroy spatial learning and memory abilities of
offspring, the mechanism of
which is related to been improving maternal plasma corticosterone and reduced hippocampal BDNF, Arc of offspring rats. However, the exact mechanism involved remains to be further elucidated.
Acknowledgement The authors declare that no competing interests exist. This work was supported by Grants from Youth Science Fund of Xinjiang Uighur Autonomous Region (2013211B50). The funding agency did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Reference Abdul Aziz, N.H., Kendall, D.A., and Pardon, M.C. 2012. Prenatal exposure to chronic mild stress increases corticosterone levels in the amniotic fluid and induces cognitive deficits in female offspring, improved by treatment with the antidepressant drug amitriptyline. Behavioural brain research 231, 29-39. Aguilera, G. 2011. HPA axis responsiveness to stress: implications for healthy aging. Experimental gerontology 46, 90-95. Aisa, B., Elizalde, N., Tordera, R., Lasheras, B., Del Rio, J., and Ramirez, M.J. 2009. Effects of 19
neonatal stress on markers of synaptic plasticity in the hippocampus: implications for spatial memory. Hippocampus 19, 1222-1231. Carvalho-Netto, E.F., Myers, B., Jones, K., Solomon, M.B., and Herman, J.P. 2011. Sex differences in synaptic plasticity in stress-responsive brain regions following chronic variable stress. Physiology & behavior 104, 242-247. de Quervain, D.J., Aerni, A., Schelling, G., and Roozendaal, B. 2009. Glucocorticoids and the regulation of memory in health and disease. Frontiers in neuroendocrinology 30, 358-370. Dole, N., Savitz, D.A., Hertz-Picciotto, I., Siega-Riz, A.M., McMahon, M.J., and Buekens, P. 2003. Maternal stress and preterm birth. American journal of epidemiology 157, 14-24. Feldt, K., Raikkonen, K., Pyhala, R., Jones, A., Phillips, D.I., Eriksson, J.G., Pesonen, A.K., Heinonen, K., Jarvenpaa, A.L., Strandberg, T.E., et al. 2011. Body size at birth and cardiovascular response to and recovery from mental stress in children. Journal of human hypertension 25, 231-240. Foster, C.J., Garber, J., and Durlak, J.A. 2008. Current and past maternal depression, maternal interaction behaviors, and children's externalizing and internalizing symptoms. Journal of abnormal child psychology 36, 527-537. Fumagalli, F., Franchi, C., Caffino, L., Racagni, G., Riva, M.A., and Cervo, L. 2009. Single session of cocaine intravenous self-administration shapes goal-oriented behaviours and up-regulates Arc mRNA levels in rat medial prefrontal cortex. The international journal of neuropsychopharmacology / official scientific journal of the Collegium Internationale Neuropsychopharmacologicum (CINP) 12, 423-429. Gale, C.R., and Martyn, C.N. 2004. Birth weight and later risk of depression in a national birth cohort. Br J Psychiatry 184, 28-33. Gronli, J., Bramham, C., Murison, R., Kanhema, T., Fiske, E., Bjorvatn, B., Ursin, R., and Portas, C.M. 2006. Chronic mild stress inhibits BDNF protein expression and CREB activation in the dentate gyrus but not in the hippocampus proper. Pharmacology, biochemistry, and behavior 85, 842-849. Hearing, M.C., Schochet, T.L., See, R.E., and McGinty, J.F. 2010. Context-driven cocaine-seeking in abstinent rats increases activity-regulated gene expression in the basolateral amygdala and dorsal hippocampus differentially following short and long periods of abstinence. Neuroscience 170, 570-579. Hsu, A.A., von Elten, K., Chan, D., Flynn, T., Walker, K., Barnhill, J., Naun, C., Pedersen, A.M., Ponaman, M., Fredericks, G.J., et al. 2012. Characterization of the cortisol stress response to sedation and anesthesia in children. The Journal of clinical endocrinology and metabolism 97, E1830-1835. Inberg, S., Elkobi, A., Edri, E., and Rosenblum, K. 2013. Taste familiarity is inversely correlated with Arc/Arg3.1 hemispheric lateralization. J Neurosci 33, 11734-11743. Johnson, B.N., and Yamamoto, B.K. 2010. Chronic stress enhances the corticosterone response and neurotoxicity to +3,4-methylenedioxymethamphetamine (MDMA): the role of ambient temperature. The Journal of pharmacology and experimental therapeutics 335, 180-189. Kajantie, E., Osmond, C., Barker, D.J., Forsen, T., Phillips, D.I., and Eriksson, J.G. 2005. Size at birth as a predictor of mortality in hood: a follow-up of 350 000 person-years. International journal of epidemiology 34, 655-663. Kapoor, A., Dunn, E., Kostaki, A., Andrews, M.H., and Matthews, S.G. 2006. Fetal programming of hypothalamo-pituitary-adrenal function: prenatal stress and glucocorticoids. The Journal of physiology 572, 31-44. Khalil, R., King, M.A., and Soliman, M.R. 2005. Testosterone reverses ethanol-induced deficit in spatial reference memory in castrated rats. Pharmacology 75, 87-92. Koenig, J.I., Kirkpatrick, B., and Lee, P. 2002. Glucocorticoid hormones and early brain development
20
in schizophrenia. Neuropsychopharmacology 27, 309-318. Kosten, T.A., Karanian, D.A., Yeh, J., Haile, C.N., Kim, J.J., Kehoe, P., and Bahr, B.A. 2007. Memory impairments and hippocampal modifications in
rats with neonatal isolation stress experience.
Neurobiology of learning and memory 88, 167-176. Kozisek, M.E., Middlemas, D., and Bylund, D.B. 2008. Brain-derived neurotrophic factor and its receptor tropomyosin-related kinase B in the mechanism of action of antidepressant therapies. Pharmacology & therapeutics 117, 30-51. Kramer, M.S., Lydon, J., Seguin, L., Goulet, L., Kahn, S.R., McNamara, H., Genest, J., Dassa, C., Chen, M.F., Sharma, S., et al. 2009. Stress pathways to spontaneous preterm birth: the role of stressors, psychological distress, and stress hormones. American journal of epidemiology 169, 1319-1326. Le Moal, M. 2007. Historical approach and evolution of the stress concept: a personal account. Psychoneuroendocrinology 32 Suppl 1, S3-9. Leung, E., Tasker, S.L., Atkinson, L., Vaillancourt, T., Schulkin, J., and Schmidt, L.A. 2010. Perceived maternal stress during pregnancy and its relation to infant stress reactivity at 2 days and 10 months of postnatal life. Clinical pediatrics 49, 158-165. Li, M., Hou, Y.Y., Lu, B., Chen, J., Chi, Z.Q., and Liu, J.G. 2009. Expression pattern of neural synaptic plasticity marker-Arc in different brain regions induced by conditioned drug withdrawal from acute morphine-dependent rats. Acta pharmacologica Sinica 30, 282-290. Li, M., Li, X., Zhang, X., Ren, J., Jiang, H., Wang, Y., Ma, Y., and Cheng, W. 2014. Effects of prenatal chronic mild stress exposure on hippocampal cell proliferation, expression of GSK-3alpha, beta and NR2B in
offspring during fear extinction in rats. Int J Dev Neurosci 35, 16-24.
Li, Z.Y., Zheng, X.Y., Gao, X.X., Zhou, Y.Z., Sun, H.F., Zhang, L.Z., Guo, X.Q., Du, G.H., and Qin, X.M. 2010. Study of plasma metabolic profiling and biomarkers of chronic unpredictable mild stress rats based on gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom 24, 3539-3546. Liu, X.H., Qian, L.J., Gong, J.B., Shen, J., Zhang, X.M., and Qian, X.H. 2004. Proteomic analysis of mitochondrial proteins in cardiomyocytes from chronic stressed rat. Proteomics 4, 3167-3176. Lobel, M., Cannella, D.L., Graham, J.E., DeVincent, C., Schneider, J., and Meyer, B.A. 2008. Pregnancy-specific stress, prenatal health behaviors, and birth outcomes. Health Psychol 27, 604-615. Maioli, S., Puerta, E., Merino-Serrais, P., Fusari, L., Gil-Bea, F., Rimondini, R., and Cedazo-Minguez, A. 2012. Combination of apolipoprotein E4 and high carbohydrate diet reduces hippocampal BDNF and arc levels and impairs memory in young mice. J Alzheimers Dis 32, 341-355. McLaughlin, K.J., Gomez, J.L., Baran, S.E., and Conrad, C.D. 2007. The effects of chronic stress on hippocampal morphology and function: an evaluation of chronic restraint paradigms. Brain Res 1161, 56-64. Moron, J.A., Gullapalli, S., Taylor, C., Gupta, A., Gomes, I., and Devi, L.A. 2010. Modulation of opiate-related signaling molecules in morphine-dependent conditioned behavior: conditioned place preference to morphine induces CREB phosphorylation. Neuropsychopharmacology 35, 955-966. Obel, C., Hedegaard, M., Henriksen, T.B., Secher, N.J., Olsen, J., and Levine, S. 2005. Stress and salivary cortisol during pregnancy. Psychoneuroendocrinology 30, 647-656. Prut, L., and Belzung, C. 2003. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. European journal of pharmacology 463, 3-33. Rizk, P., Salazar, J., Raisman-Vozari, R., Marien, M., Ruberg, M., Colpaert, F., and Debeir, T. 2006. The alpha2-adrenoceptor antagonist dexefaroxan enhances hippocampal neurogenesis by increasing the survival and differentiation of new granule cells. Neuropsychopharmacology 31, 1146-1157.
21
Salomon, S., Bejar, C., Schorer-Apelbaum, D., and Weinstock, M. 2011. Corticosterone mediates some but not other behavioural changes induced by prenatal stress in rats. Journal of neuroendocrinology 23, 118-128. Shepherd, J.D., and Bear, M.F. 2011. New views of Arc, a master regulator of synaptic plasticity. Nature neuroscience 14, 279-284. Simon, P., Dupuis, R., and Costentin, J. 1994. Thigmotaxis as an index of anxiety in mice. Influence of dopaminergic transmissions. Behavioural brain research 61, 59-64. Sirianni, R.W., Olausson, P., Chiu, A.S., Taylor, J.R., and Saltzman, W.M. 2010. The behavioral and biochemical effects of BDNF containing polymers implanted in the hippocampus of rats. Brain Res 1321, 40-50. Steward, O., and Worley, P. 2002. Local synthesis of proteins at synaptic sites on dendrites: role in synaptic plasticity and memory consolidation? Neurobiology of learning and memory 78, 508-527. Waddell, S., and Jenkins, J.R. 1995. arg3+, a new selection marker system for Schizosaccharomyces pombe: application of ura4+ as a removable integration marker. Nucleic acids research 23, 1836-1837. Wadhwa, P.D. 2005. Psychoneuroendocrine processes in human pregnancy influence fetal development and health. Psychoneuroendocrinology 30, 724-743. Weinstock, M. 2005. The potential influence of maternal stress hormones on development and mental health of the offspring. Brain, behavior, and immunity 19, 296-308. Willner, P. 1997. Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology 134, 319-329. Yang, J., Pei, Y., Pan, Y.L., Jia, J., Shi, C., Yu, Y., Deng, J.H., Li, B., Gong, X.L., Wang, X., et al. 2013. Enhanced antidepressant-like effects of electroacupuncture combined with citalopram in a rat model of depression. Evid Based Complement Alternat Med 2013, 107380. Yu, I.T., Lee, S.H., Lee, Y.S., and Son, H. 2004. Differential effects of corticosterone and dexamethasone on hippocampal neurogenesis in vitro. Biochemical and biophysical research communications 317, 484-490. Zhong, N., Scearce-Levie, K., Ramaswamy, G., and Weisgraber, K.H. 2008. Apolipoprotein E4 domain interaction: synaptic and cognitive deficits in mice. Alzheimers Dement 4, 179-192.
22
Figure Captions Figure 1 The comparison of plasma corticosterone level between PS and PC group The figure shows that changes in plasma corticosterone level of PS were noted after stress model is established successfully. Data were analyzed using repeated measurement analysis of variance. Each column represents mean ± SD. Number of animals in each group = 10. *P < 0.05 vs. PC group.
Figure 2 The comparison of behaviors in open-field test between PS and PC groups At regular time intervals throughout the 21 days of stress, differences in horizontal movement (A) and vertical movement (B) were observed between CUMS and control groups. Data were analyzed using repeated measurement analysis of variance. Each column represents mean ± SD. Number of animals in each group=10. *P < 0.05 vs. PC group.
23
Figure 3 The comparison on behaviors of liquid consumption test between PS and PC group At regular time intervals throughout the 21 days of stress, differences in pure water consumption (A), sugar water consumption (B), total liquid consumption (C) and sucrose preference (D) were detected between PS and PC groups. Data were analyzed using repeated measurement analysis of variance. Each column represents mean ± SD. Number of animals in each group = 10. *P < 0.05 vs. PC group.
24
Figure 4 The comparison on the body weight between PS and PC groups At regular time intervals throughout the 21 days of stress, differences in maternal body weight were detected between PS and PC groups. The growth rate of maternal body weight in PS was lower than PC group. Data were analyzed using repeated measurement analysis of variance. Number of animals in each group = 10. Each column represents mean ± SD. *P < 0.05 vs. PC group.
25
Figure 5 The comparison on the basic situation of reproduction between PS and PC group At regular time intervals throughout the 21 days of stress in pregnancy, observations for differences in the number of offspring (A), days of pregnancy (B) and the proportion of male and female (C) were made between PS and PC groups. The number of offspring and days of pregnancy in PS group was less than the PC group. Data were analyzed using student’s t test. Each column represents mean ± SD. Number of animals in each group = 10. *P < 0.05 vs. PC group.
26
Figure 6 The comparison between PS offspring and PC offspring in body weight and plasma corticosterone level Differences in body weight and plasma corticosterone level were detected between PS offspring and PC offspring. The growth rate of offspring in PS was lower than PC group. The plasma corticosterone level of PS offspring was higher than that of PC offspring. On different postnatal days, data were respectively analyzed using student’s t test. Each column represents mean ± SD. Number of animals in each offspring group = 16 (50% male, 50% female) *P < 0.05 vs. PC offspring.
27
Figure 7 Comparison in escapes latency and crossing platform between PS and PC offspring groups Differences in escapes latency and crossing platform were detected between PS and PC offspring from PND 42. The rate of increase of escapes latency and crossing platform in PS group was lower than PC group. Data were analyzed using repeated measurement analysis of variance. Each column represents mean ± SD. Number of animals in each offspring group = 16 (50% male, 50% female) *P < 0.05 vs. PC offspring.
28
Figure 8 Histological section of hippocampus Representative photomicrographs of the hippocampal CA1 area (×200, A), hippocampal neurons (×1 0000, B) and Hippocampal synaptic cell (×5 0000, C)
Figure 9 The effects of maternal stress during pregnancy on the BDNF and Arc expression in the offspring’s hippocampus Differences in BDNF and Arc 29
expression in the hippocampus were detected between PS offspring and PC offspring. The BDNF mRNA, protein and Arc protein expression of PS offspring were lower than that of PC offspring. At different postnatal day, data were respectively analyzed using student’s t test. Each column represents mean ± SD. Number of animals in each offspring group = 16 (male, female in half). *P < 0.05 vs. PC offspring.
30
Table 1 Correlations between learning and memory of offspring and the determination results of their mothers PS PS offspring Plasma corticosterone
Horizontal movement
Sucrose preference
Escape latency
0.556*
-0.464*
-0.626*
Crossing platform
-0.441*
0.676*
0.630*
* P<0.05 Table 2 Correlations between learning and memory and protein expression of offspring Parameters
BDNF
Arc
Escape latency
-0.589*
-0.421*
Crossing platform
0.556*
0.458*
* P<0.05
31
S1382-6689(16)30089-8 http://dx.doi.org/doi:10.1016/j.etap.2016.04.012 ENVTOX 2498
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
9-11-2015 20-4-2016 23-4-2016
Please cite this article as: Guan, Su-zhen, Ning, Li, Tao, Ning, Lian, Yu-long, Liu, Ji-wen, Ng, Tzi Bun, Effects of maternal stress during pregnancy on learning and memory via hippocampal BDNF, Arc (Arg3.1) expression in offspring.Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2016.04.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of maternal stress during pregnancy on learning and memory via hippocampal BDNF, Arc (Arg3.1) expression in offspring
Su-zhen Guan1, Li Ning2, Ning Tao2, Yu-long Lian3, Ji-wen Liu2,*, Tzi Bun Ng4,*
1
Department of Social Medicine, College of Public Health, Xinjiang Medical
University, Urumqi 830011, China; 2
Department of Occupational Health and Environmental Health, College of Public
Health, Xinjiang Medical University, Urumqi 830011, China; 3
Department of Occupational Health and Environmental Health, College of Public
Health, College of Medical, Nantong University, Jiangsu 226000, China; 4
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of
Hong Kong, Hong Kong.
*
Corresponding authors:
Ji-wen Liu Tel no.: 86-9914365004 Fax no.: 86-9914365004 Email: [email protected] Tzi Bun Ng Tel no.: 852-39436872 Fax no.: 852-26035123 Email: [email protected]
1
Graphical abstract
Research Highlights Chronic stress in pregnancy affected spatial learning and memory of the offspring Plasma corticosterone level was increased The expression of Arc protein and BDNF protein and mRNA in offspring was attenuated
2
ABSTRACT The intrauterine environment has a significant long-term impact on individual’s life, this study was designed to investigate the effect of stress during pregnancy on offspring’s learning and memory abilities and analyze its mechanisms from the expression of BDNF and Arc in the hippocampus of the offspring. A rat model of maternal chronic stress during pregnancy was mating from 3rd day during been subjecting to chronic unpredictable mild stress (CUMS). The body weights and behavioral changes were recorded, and plasma corticosterone levels were determined by radioimmunoassay. The learning and memory abilities of the offspring were measured by Morris water maze testing from PND 42. The expression of hippocampal BDNF and Arc mRNA and protein were respectively measured using RT-PCR and Western blotting. Results indicated that an elevation was observed in the plasma corticosterone level of rat model of maternal chronic stress during pregnancy, a reduction in the crossing and rearing movement times and the preference for sucrose. The body weight of maternal stress’s offspring was lower than the control group, and the plasma corticosterone level was increased. Chronic stress during pregnancy had a significant impact on the spatial learning and memory of the offspring. The expression of BDNF mRNA and protein, Arc protein in offspring of maternal stress during pregnancy was attenuated and some relationships existed between these parameters. Collectively, these findings disclose that long-time maternal stress during pregnancy could destroy spatial learning and memory abilities of the offspring, the mechanism of which is related to been improving maternal plasma corticosterone and reduced hippocampal BDNF, Arc of offspring rats. Keywords: Maternal chronic stress; pregnancy; learning and memory; BDNF; Arc /Arg3.1; offspring
3
1. Introduction In the daily life, people often encounter different kinds of psychosocial stress, even at some specific physiological stages. It is difficult to avoid acute or chronic psychological stress, which may have a significant impact on health. Some women also experience daily stress during pregnancy, these stresses include depression, anxiety, anger, day-to-day challenges, sudden change of environment, social isolation, and so on. Maternal stress during pregnancy has been implicated as one of the risk factors for development in the offspring (Kramer et al., 2009; Le Moal, 2007). A large number of epidemiological evidence has indicated that a variety adverse pregnancy outcomes appear during maternal stress: the higher of the maternal plasma cortisol levels, birth rate of low birth weight babies and preterm birth (Gale and Martyn, 2004; Kajantie et al., 2005). It may also cause a rise in disruption of sleep patterns, reduction of deep sleep time, abnormality in sleep time, and prolongation of crying time (Leung et al., 2010). In addition, it can also have an effect on children's later growth and lead to weaker attentiveness in school-age children, emotional and behavioral problems and poor learning and memory abilities (Lobel et al., 2008). Recent studies have indicated that stress experiences of fatal stage affect synaptic plasticity and neurogenesis in specific areas of the central nervous system, particularly in the hippocampus (Aisa et al., 2009), which is part of the limbic system in brain and has a major role in cognition and mood regulation. Some studies have shown that stress during pregnancy inhibits cell proliferation in the hippocampus of offspring which causes impairment of learning and memory (Aguilera, 2011). The brain-derived neurotrophic factor (BDNF), which is directly involved in many physiological aspects of central nervous system such as neurite growth and neuronal survival (Kozisek et al., 2008), and plays a key role in learning and memory. Other data suggest that BDNF is also involved in both short- and long-term plasticity of glutamatergic synapses. BDNF signaling enhances synaptic maturation and increases synaptic density in the hippocampus, the synthesis and secretion of BDNF in the continue to be regulated by activity (Gronli et al., 2006). Another important neural basis of learning and memory is immediate early genes (IEGs), which is a kind of 4
encoding transcription factor protooncogene. A new kind of IEGs, activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), has recently gained a significant amount of attention. When the synaptic activity was increased, the expression of Arc/Arg3.1 in hippocampal neuron dendrites was significantly increased after stress, which was directly involved in synaptic plasticity and memory consolidation (Shepherd and Bear, 2011). The intrauterine environment has a significant long-term impact on individual’s life, it was hypothesized that maternal stress during pregnancy will have a negative effect on the offspring’s learning and memory, at the same time, exposure of the fetus in higher levels of corticosterone (It is a glucocorticoid in rodents and the primary end product of the hypothalamic– pituitary– adrenal axis) and the changes of expression of hippocampal BDNF and Arc maybe is the important reason for learning and memory. Therefore, this study was designed to investigate the effect of stress during pregnancy on
offspring’s learning and memory and analyze its mechanisms from
the expression of BDNF and Arc in the hippocampus.
2. Materials and methods 2.1. Animals Twenty female
Wistar rats weighing 240~270g and fifteen male Wistar rats
weighing 300~350g and sexually mature [all supplied by the Animal Laboratory Center of Xinjiang Medical University, Urumqi, Xinjiang, China; experimental animal certificate number: SCXK (new), 2011-000.], which were randomly allocated into seven cages (5 rats in each cage, male and female apart) after acclimatization for a week. All rats were maintained under standard laboratory conditions (12 h light/dark cycle, temperature 21-23℃, relative humidity 45-65%, and food and water ad libitum) during this week. 2.2. Chronic unpredictable mild stress (CUMS ) procedure The CUMS procedure followed a previously described method (Willner, 1997) with minor modifications. Chronic unpredictable mild stress comprised exposure to the following stressors in a random sequence everyday for 3 weeks: food deprivation 5
for 24 h; water deprivation for 24 h; cage tilt (45◦,7 h); noise housing (1500 Hz, 92 db, for 1 h); behavioral restriction for 4 h; forced swimming for 1 h in a 31℃ water bath; squeezing tail for 1 min; shaking stress ( 30 min); hot stress in an oven (42℃, 5 min); soiled cage (200 ml of water poured into the bedding, 8 h). One of the ten different stressors was randomly administered each day. During the process, the model rats were moved into another room (light intensity and temperature of two rooms were basically the same), then back to the room after the stimulation. 2.3. The treatments of maternal stress rat during pregnancy The maternal stress rat during pregnancy was mating from 3rd day during subjected to CUMS (as shown in the figure below). Twenty female Wistar rats were randomly allocated into two groups (10 rats per group), namely, maternal chronic stress rat during pregnancy model group (PS group) and control group (PC group). Before gestation, PS group was mated with a male in one cage, two rats in PC group were mated with a male rat in one cage. All female rats were examined every morning and pregnancy was confirmed by sperm positivity. Then male and female rats were separated after female conception, which was designated gestational day 0 (GD 0). After gestation, there were also 10 rats in each group. PC rats were housed with 5 in each cage (1 per cage after GD18), while PS rats were housed individually (1 per cage). Every stress factors didn’t suspend during mating. All rats were maintained under standard laboratory conditions.
Fig. Experimental procedure
6
2.4. Measurement of CUMS model 2.4.1. Measurement of plasma corticosterone level Venous blood samples (1 ml) were collected from the rats on the day before stress (Baseline) and then on the 1st, 7th, and 14th. Plasma was used for determination of plasma corticosterone level. Blood samples were centrifuged (3000 rpm for 20 min at 4℃), and the plasma obtained was stored at −35℃. Corticosterone was measured using a radioimmunoassay (RIA) kit in accordance with the manufacturer’s instructions (Coat-A-Count, Diagnostics Products Corporation). The intra-assay variability of the RIA ranged between 3.2% and 4.7%. The plasma corticosterone levels were derived from the determined cortisol values using the following conversion formula: Corticosterone concentration=Cortisol concentration ×50 (Liu et al., 2004). 2.4.2. Open-field test An open-field method was used to conduct praxeological scoring in the two groups of rats. The open-field device was made of opaque materials with a 80 cm × 80 cm square, located at the bottom, which was divided equally into 25 equilateral squares. Surrounding it was a wall with a height of 40 cm. The rat was put in the central square, and the number of squares the rat traversed in 3 min was recorded (only the squares on which the rat landed with four legs could be numbered as the score of horizontal activity) and the duration of standing on hind limbs was noted. Each rat was measured once for 3 min. A score was given by each of the two observers and the average value was taken. The percentage time spent in this central zone is considered indicative of exploratory behavior and may reflect a decrease in anxiety, although this parameter is not sensitive to all anxiolytics and may not model certain features of anxiety disorders (Li et al., 2010). The results reflect mean values of daily tests over three days. 2.4.3. Sucrose preference test In the sucrose preference test, the animals were allowed to consume water and a 1% sucrose solution for 1 h after food and water deprivation for 20 h, following 48 h of exposure to both water and sucrose solution. The positions of the two bottles (right/left) were varied randomly across animals and were reversed after 30 min. 7
Sucrose preference was calculated according to the following ratio: sucrose preference (%) = [sucrose intake (g)/sucrose intake (g) + water intake(g)]×100% (Prut and Belzung, 2003). The results reflect mean values of daily tests over three days. 2.5. Allocation of the offspring into groups The day on which the offspring were born was designated as postnatal day 0 (PND 0). The offspring were weaned from their mothers on PND 21, and male and female offspring separated. They remained undisturbed and were allocated to two groups, consisting of 8 male vs. 8 female offspring with PS, and 8 vs. 8 with PC, respectively. The 16 offspring from PS were called PS offspring, and offspring from PC were called PC offspring. Offsprings were housed in cages with up to 8 rats. 2.6. Measurement of offspring’s learning and memory abilities--Morris water maze testing(MWM) 2.6.1. Testing room and apparatus The water maze apparatus consisted of a circular pool (1.54 m diameter) made of stainless steel. The pool was filled with water (25±1℃) that was made opaque by the addition of non-sugar milk powder. During MWM training, an escape platform (20 cm diameter) made of stainless steel with a grooved surface was submerged 5 cm under the water level. All tests were carried out in the same experimental room and the MWM testing area was isolated from the operator and computer by a white curtain. Dark boards of different shapes provided landmarks in the testing room. Three days prior to the commencement of the acquisition phase, offspring underwent training to acclimatize them to the testing room and pool. The platform was placed in the centre of the pool and offspring were placed on the platform three times for 15 s. 2.6.2. Acquisition phase and reversal task All animals were tested in MWM started from PND 42 for five consecutive days. On each day, animals received two sessions of testing, at 10:00～12:00 am and 18:00～20:00 pm. Each session consisted of four trials with an inter-trial period of 10 min. For each trial, the animal was released from 1 of the 4 cardinal compass points (N, S, E, W). The animal was allowed a maximum of 120 s to locate and mount the escape platform with a post-trial timeout of 15 s on the platform. Escape latency (EL), 8
the time of locating the platform was our record time. The last EL was the average of EL from four different quadrants respectively in 5 days. After the test, the platform was removed, the offspring were put into the water from the same entry to nod to the wall, allowed to swim for 2 min. Closed the window and the curtains, fixed facilities of the room, in order to make sure that there was not any clue to mark the position of platform. Need to determine an effective positioning capability of the fixed structure within and outside the maze as a clue. Another index, the number of crossing platform, refers to the number of offspring shuttling the platform through the original position after removing the platform in a certain period of time. 2.7. Tissue collection from offspring For all subjects, the brain was removed after determination of learning and memory ability. Following intraperitoneal injection of chloral hydrate anesthesia, the hippocampus was excised from the brain, placed on a freezing aluminum dissection stage, bisected midway between the septal and temporal poles. One half of the left hippocampus was fixed with 4% formalin solution for HE sliced pathology (the change of structure of hippocampal CA1 area), and half of them were fixed with glutaraldehyde for electron microscopy. Then rapidly put right hippocampus frozen in liquid nitrogen and dissected on ice-cold glass plates, frozen on dry ice and stored at -80℃. The principle of using 50% male and 50% female was followed during the process. 2.8. Quantitative real-time RT-PCR Total RNA was extracted from 50-100 mg hippocampus according to the instructions of “RNA-BeeTM isolation of RNA” kit (TRIzol® Reagent, Life Technologies, USA). Extracted RNA was quantified by Nucleic acid protein quantitative instrument. Samples with the A260/280 ratio between 1.8-2.0 were used. Briefly, after homogenization of the tissue in reaction mixture containing RNA-Bee and chloroform and centrifugation at 12 000×g for 15 min at 4℃, the extracted RNA
9
in the aqueous phase was obtained. 200 µl of this aqueous phase was mixed with the same volume of isopropanol and after centrifugation at 12 000×g for 15 min at 4℃, RNA was precipitated and formed a pellet. The pellet was washed with 75% ethanol (by vortexing and centrifugation at 7 500×g for 5 min at 4℃) and then ethanol was removed and the pellet of RNA was allowed to dry, and then solubilized in 25 µl DEPC water. RNA concentration and purity were evaluated by spectrometry by optical density (OD) measurements at 260 -280 nm. A two-step reverse-transcription quantitative PCR (RT-qPCR) assay was performed, and both reactions [reverse transcription (RT) and quantitative polymerase chain reaction (qPCR)] were performed in a Chromo 4 Real Time PCR Instrument (MJ Research, USA). cDNA was generated from 600 ng of total RNA in a total volume of 40 μl using a cDNA synthesis kit (ReverttAid First Strand cDNA Synthesis Kit, USA). PCR was performed using the SYBR® Select Master Mix reagent for performing realtime PCR assays for the genes encoding BDNF and Arc. RT-PCR primers used were as follows: BDNF (NM_001270630.1): forward, 5'- AGGCACT GGAACTCGCAATG-3', reverse, 5'-AAGGGCCCGAACATACGATT-3'; Arc (NM_019361.1): forward, 5'-CCCATCTATGAGGGTTACGC-3', reverse, 5'-TTTAATGTCACGCACGATTT C-3'; β-actin (NM_031144.3): forward, 5'- CCCATCTATGAGGG TTACGC-3', reverse, 5'- TTTAATGTCACGCACGA TTTC-3'. The thermal cycling conditions were as follows: 2 min at 50℃ and 10 min at 95℃ followed by 40 cycles of 95℃ for 15 s and 60℃ for 1 min. The threshold value (Ct) for each sample was set in the exponential phase of PCR, and the ΔΔCt method was used for data analysis. β-action was used as reference gene. The experiment was performed in triplicate. 2.9. Western blotting Following tissue homogenization, protein concentration was assayed using a bicinchoninic acid (BCA) test (Beijing Tiangen, China). Punches were sonicated in 120-150 μl ice-cold RIPA buffer containing 50 mM Tris-HCl (pH 8.8), 150 mM NaCl, 1% NP-40 and 0.1% SDS (Beijing Applygen Technologies Inc, Beijing, China). The homogenate was then centrifuged at 12 000 g for 5 min, and the supernatant saved for analysis. Protein concentrations were determined using the BCA assay (Pierce, 10
Rockford, IL). Sample buffer was immediately added to the homogenates, and the samples were boiled for 5 min. Protein extracts (30 μg) were then electrophoresed in 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Blots were blocked in TBS buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.05% Tween 20) with 5% dry milk and incubated with an anti-Arc/Arg3.1 antibody (1:100; Santa Cruz Biotechnology), anti-BDNF antibody (1:200; Santa Cruz Biotechnology). Blots were then incubated with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:10 000; Santa Cruz Biotechnology) for 1 h at room temperature and developed using the West Dura chemiluminescent substrate (Pierce Laboratories). Densitometry was determined based on band intensity, and relative protein expression was quantified by densitometry using the Total Lab 2.01 analysis system (Phoretix, UK). To control for inconsistencies in loading, optical densities were normalized to β-action protein expression. Data for treated animals were normalized to the average value of the naive controls. 2.10. Statistical analysis Statistical analysis of all data, expressed as mean ± SD, was performed using the Statistical Package for the Social Sciences 13.0, and all graphs were constructed in GraphPad Prism 5.0. Differences between the means of maternal data in plasma corticosterone level, open-field test, sucrose preference test and the performance of offspring in MWM were analyzed using repeated measurement analysis of variance, LSD-t test to make multiple comparison at the different time. All RT-PCR and Western blotting data were analysed using student’s t test for comparison between two offspring groups. Bivariate correlations were performed using Spearman correlations. P value less than 0.05 was considered to be statistically significant.
3. Results 3.1. CUMS elevated maternal plasma corticosterone level The repeated measurement analysis of variance showed that chronic stress had a significant impact on the circulatory corticosterone level (F=14.996, P=0.001). Corticosterone level of the PS group drastically changed with stress time (F=64.607, 11
P<0.001). At the same time, there was an interactive relationship between time and stress factors (F=8.144, P=0.001). LSD-t test revealed that plasma corticosterone level of the PS group rose to the peak value which was higher than that of the PC group following exposure to stress for 7 days (t=9.378, P<0.001). However, the plasma corticosterone level of the PS group declined after exposure to stress for 14 days, but still remained higher than that of the PC group (t=4.437, P<0.001), indicating that the PS group was in a stressful state. Plasma corticosterone levels did not reach statistical significance between rats in the PC group and the PS group at the time of the baseline (t=1.128, P=0.130) and after exposure to stress for 1 day (t=0.738, P=0.235) (Figure1).
3.2. CUMS reduced horizontal and vertical movements in PS rats The repeated measurement analysis of variance revealed that chronic stress significantly affected the horizontal and vertical movements in the PS group (F =12.347, 8.541; all P<0.001). After exposure to stress for 21 days, both horizontal movement (Figure 2A) and vertical movement (Figure 2B) in the PS group were lower than those of the PC group (t day21=3.828, 3.535; all P<0.001 ) by LSD-t test. However, no significant differences between the PS group and the PC group were observed at baseline and after exposure to stress for 1, 7, 14 day (t P=0.218, 0.310. t 0.132. t
day14
day1=0.313,
0.288; P=0.379, 0.388; t
day7
day0=0.796,
0.497;
=0.827, 1.565; P=0.417,
=1.651, 1.777; P=0.113, 0.089.) (Figure 2). The reduction of horizontal
movements reflects that animal's activity reduced, the vertical motion reflects less curiosity about the new environment in which the animals was subjected to.
3.3. CUMS decreased Liquid consumption in PS rats The repeated measurement analysis of variance revealed that chronic stress significantly affected sugar water consumption, total liquid consumption and 1% sucrose preference in the PS rats (F=11.819, 6.966, 10.548; all P<0.001). It showed that stress affected fluid consumption index of rats during pregnancy. Regarding the baseline liquid consumption index, there was only a slight difference between the PS 12
and PC rats (P>0.05). A comparison of each point in time revealed that after 1 day of stress, sugar water consumption underwent a significant decline among rats in the PS group and the PC group by LSD-t test (all P<0.05). Following exposure to stress for 7, 14 and 21 day, it was manifested that sucrose-intake of the PS rats reduced compared with PC rats by LSD-t test (all P<0.05). Similarly, pure water consumption of the model group was higher than the PC group by LSD-t test (all P<0.05) (Figure 3).
3.4. CUMS reduced the rate of increase of maternal body weight The repeated measurement analysis of variance revealed that chronic stress significantly affected the rate of increase of maternal body weight in the PS rats (F=4.854, P=0.044), indicating that stress had a negative effect on rat weight gain during pregnancy. Along with the prolongation of the stress time, the rate of weight increase in rats of the model group was lower than the control group (F=85.191, P<0.001). Following exposure to stress for 7, 14 and 21 day, the body weight of PC rats was higher than rats in the PS group by LSD-t test (all P<0.05). However, no significant differences between the PS group and the PC group were observed at baseline and after stress for 1 day (P >0.05) (Figure 4).
3.5. Reproductive ability was impaired in PS rats There was statistically significant difference between the PS group and the PC group in the number of offspring and days of pregnancy (t=3.145, P=0.012; t=2.186, P=0.042) using student’s t test. The number of offspring (Figure 5A) and days of pregnancy (Figure 5B) in PS group was less than those of the PC group, indicating that maternal stress during pregnancy rat adversely influenced the reproductive ability of them. No significant differences were observed in sex distribution between two groups (t=0.798, P=0.440) (Figure 5C) (Figure 5).
3.6. Body weight and plasma corticosterone level of offspring changed after maternal stress during pregnancy On PND 28 and 42, the weight of offspring in PS group were lower than PC 13
(t=4.391, P<0.001; t=1.966, P=0.001) using student’s t test, showing that maternal stress during pregnancy affected the growth rate of offspring as seen in the body weight (Figure 6A). Using student’s t test, the plasma corticosterone level in PS offspring was higher than that in PC offspring on PND 28 and 42 (t=-2.251, P=0.035; t=-3.767, P=0.001), indicating that plasma corticosterone level in offspring of maternal stress during pregnancy was elevated (Figure 6B) (Figure 6).
3.7. Spatial learning and memory of
offspring in MWM changed after maternal
stress during pregnancy The repeated measurement analysis of variance showed that chronic stress during pregnancy had a significant impact on the escape latency of offspring from PND 42 (F=7.578，P<0.001), the escape latency of PS offspring were higher than that of PC. The escape latency of the PS offspring drastically changed with stress time (F=64.682，P<0.001). At the same time, there was no interactive relationship between time and stress factors (F=1.444，P=0.228). LSD-t test revealed the escape latency of the PS offspring group was higher than that of the PC offspring following measured 1, 4 and 5 days (Figure 7A) (tday1=2.598, P=0.004; tday4=2.638, P=0.003; tday5=8.755, P<0.001). However, escape latency did not differ between offspring in the PC group and the PS group at the time of measuring 2 and 3 days (tday2=0.569, P=0.287; tday3=0.309, P=0.380). The number of crossing platform of PS offspring was lower than that of PC by independent sample t-test (t=2.919, P=0.003) (Figure 7B). The results suggested that maternal stress during pregnancy had a significant impact on the spatial learning and memory of
offspring from the escape latency and crossing
platform in MWM (Figure 7).
3.8. Hippocampal structure of offspring damaged due to Maternal stress during pregnancy Observed under HE pigmentation with light microscope, each layer of hippocampus structure in PS offspring was not clear, cell morphology was irregular, 14
the gap of cell increased and arranged loosely (Figure 8A), suggested that structure of hippocampal CA1 area of offspring in maternal stress during pregnancy been changed. The results of electron microscope showed that the hippocampal neurons of PS offspring changed such as nuclear membrane invagination, reduction of nucleolar volume, nuclear vacuolated structure (Figure 8B). A series of changes in hippocampal synaptic cell were found, including granules sparsity, degranulation , the quantity of ribosome been reduced, the presynaptic membrane particles decreased in PS offspring compared with PC control group (Figure 8C). 3.9. Maternal stress during pregnancy effects on Arc and BDNF expression in offspring’ hippocampus from RT-PCR and Western blotting Determination of the extracting RNA by ultraviolet spectrophotometry showed that the ratio of A 260/A 280 lied between 1.8 ~ 2.0 and two clear bands in 28 s and 18 s were revealed after formaldehyde agarose gel electrophoresis. Strip quantity of 28 s was double that of 18 s, suggesting that RNA samples were intact (Figure 9A). Compared with PC offspring, the average expression of BDNF mRNA in PS offspring was significantly decreased (t=3.794, P=0.004) as shown by RT-PCR (Figure 9B). However, no significant differences between the PS offspring and the PC offspring were observed at the average expression of Arc (t=1.624, P=0.153). The molecular weights of BDNF and Arc were respectively 14 kDa and 50 kDa according to Western blotting results (Figure 9C). The average optical density (OD) values were displayed: statistically significant differences in hippocampal BDNF and Arc protein between PS offspring and PC offspring were detected (t=28.107, P=0.001; t=6.030, P=0.026) (Figure 9D). Compared with the PC offspring, the hippocampal BDNF and Arc protein of PS offspring were reduced (Figure 9).
3.9. Correlations of learning and memory of offspring and the determination results of their mothers
15
When all parameters of the PS offspring and their mothers were included in the correlation analysis, the escape latency of PS offspring was found to be positively correlated with the plasma corticosterone level of their mothers (r=0.556, P<0.05), and negatively correlated with horizontal movement and sucrose preference of their mother (r=-0.464, -0.626; all P<0.05). Meanwhile, crossing platform of the PS offspring was found to be positively correlated with horizontal movement and sucrose preference of the mother (r=0.676, 0.630; all P<0.05) and negatively correlated with the plasma corticosterone level of the mother (r=-0.441, P<0.05) (Table 1 ). 3.10. Correlations of learning and memory and hippocampal protein expression of offspring The escape latency of PS offspring was found to be negatively correlated with BDNF and Arc protein expression (r=-0.589, -0.421; all P<0.05). Meanwhile, crossing platform of PS offspring was found to be positively correlated with BDNF and Arc protein expression (r=0.556, 0.458; all P<0.05) (Table 2 ).
4. Discussion It is well known that many factors during pregnancy can provoke lead behavioral dysfunctions and dramatic developmental retardations. In the late 1980s, the chronic unpredictable mild stress (CUMS) model, originally developed by Paul Willner and colleagues, and based on both clinical and preclinical research regarding the etiology of stress (Willner, 1997), has been shown to mimic daily hassles and stress levels in humans. We started mating experiment during 3rd day in 21 days model period. Our experimental data showed a higher plasma corticosterone (stress hormone) level of rats in the PS group than that of PC rats, indicated that the model of maternal stress during pregnancy was established successfully. We also found that rat activity, curiosity to a new environment and reward reaction after chronic exposure to stress for 21 days during pregnancy were all diminished through behavioral and liquid consumption testing. Above all, stress during pregnancy can induce changes in the maternal neurobiological variables (Hsu et al., 2012; Yang et al., 2013). At the same time, our study clearly demonstrates that stress has an effect on 16
maternal weight gain, resulting in the increased plasma corticosterone levels of mothers, that can curb weight growth, accelerate protein decomposition and suppress protein synthesis (Abdul Aziz et al., 2012). The fetal function of the HPA axis was reprogrammed when the period of fetal exposure to high GC conditions, highly consistent with a considerable amount of related experimental results (Kapoor et al., 2006), such as, the embryo resorption, deformity, fetal growth restriction, low birth weight, and even the changes of sex ratio (Griffin et al., 2003). In human studies, maternal cortisol is known to cross the placenta and likely influence fetal development (Foster et al., 2008; Obel et al., 2005). Therefore, we found that the number of offspring and days of pregnancy in PS group was less than the PC group. Excess cortisol alters the normal functioning of the HPA axis, which may last for a lifetime (Weinstock, 2005). However, high levels of corticosteroids after stress damage the hippocampus, as it contains an elevated concentration of adrenal cortical hormone receptor in brain (McLaughlin et al., 2007) and selectively plays a role in the hippocampus, which has a pivotal function in learning and memory of rodents (Li et al., 2014; Salomon et al., 2011) . At the same time, stress can cause hippocampal neurons to change, hippocampal dendrites of pyramidal neurons shrink, which could damage the memory (Kosten et al., 2007;Carvalho-Netto et al., 2011). Our findings support the initial hypothesis drawn hippocampal structure changed in PS offspring. In particular, the present study is to assess and compare the effect of maternal stress during pregnancy on MWM performance from the offspring’s age of 42 days. Results showed that offspring whose mothers had been exposed to series of mixed stressors during pregnancy demonstrated an increased latency to find the platform and a decreased number of crossing platform, signified that maternal stress during pregnancy had a significant adverse impact on the spatial learning and memory of offspring. Similar to the findings of a more recent study, prenatal stressed rat offspring demonstrated increased latencies to find the platform in the MWM which was associated with the use of a random search strategy (Amugongo and Hlusko, 2014; Rizk et al., 2006). These findings are in line with suggestive mechanisms that glucocorticoids had been shown to affect MWM performance (Yu et al., 2004), which 17
affected the hippocampus: acute high levels facilitate hippocampal neurogenesis, synaptogenesis and dendritic remodeling, but chronically elevated levels impair these processes (Khalil et al., 2005). In rats, maternal stress during gestation increased latency in the MWM and this was associated with decreased LTP in hippocampal CA1 region slices (Kapoor et al., 2006) To determine the mechanisms underlying the effects of maternal stress during pregnancy on cognition in offspring, we focused on two molecules involved in the memory processes—BDNF and Arc/Arg3.1 (related to learning and memory). BDNF has been shown to play diverse roles in modulating the structure and function of the brain (Kozisek et al., 2008;Gronli et al., 2006). BDNF regulates dendritic and axonal morphology and affects synaptogenesis and synaptic transmission. Studying revealed that the average expression of BDNF in PS offspring was significantly decreased. The HPA axis is overactive when subjected to higher glucocorticoids, leading to BDNF down regulation, damage of mitochondria through Glu-NMDAR-NOS pathways, leading to atrophy of hippocampal neurons, apoptosis, and reduction of dentate gyrus granular cell regeneration, etc (Sirianni et al., 2010). In turn, BDNF has been reported to be an attractive candidate that translates experience-dependent neuronal activity into structural and functional changes in neuronal populations under prenatal stressful conditions (Maioli et al., 2012). Induction of immediate early genes (IEGs) is viewed as an important step in the formation of long-lasting neuro-adaptations underlying learning and memory (Inberg et al., 2013; Moron et al., 2010). Among the effector IEGs, Arc/Arg3.1 is a unique IEG that may be induced by neuronal activity and specifically trafficked and localized to recently potentiated synapses, where it may interact with structural proteins and proteins critical to synaptic plasticity (Li et al., 2009). Our present results showed that Arc/Arg3.1 protein expression of hippocampus decreased, compared with PC offspring. Zhong et al. (Zhong et al., 2008) previously reported that old mice showed impaired induction of Arc and mild hippocampus- dependent memory deficits in the MWM test. The possible reason was that higher glucocorticoids of offspring after maternal stress during pregnancy has a regulatory effect on Arc/Arg3.1 expression. 18
Moreover, chronic stress should inhibit Arc protein expression in the hippocampus, which mediates expression of AMPA receptor, the hippocampus cell swallow phenomenon appears, then can damage its own stable synaptic plasticity and impair hippocampal function. Thirdly, It is known that Arc expression is stimulated by learning-induced neuronal activity, N-methyl-D-aspartic acid receptor complexes (NMDARs) activation and LTP as well as by BDNF-TrkB signaling (Maioli et al., 2012). Apart from the HPA axis, signaling through NMDARs is critically involved in learning, memory and synaptic plasticity (Cho et al., 2009). Combined with our finding that the learning and memory of PS offspring correlated with BDNF and Arc protein expression, these findings raise the possibility that BDNF and Arc can promote synapse-specific translation during LTP production or memory formation (Fumagalli et al., 2009). As expected, the present study demonstrated that maternal stress during pregnancy could destroy spatial learning and memory abilities of
offspring, the mechanism of
which is related to been improving maternal plasma corticosterone and reduced hippocampal BDNF, Arc of offspring rats. However, the exact mechanism involved remains to be further elucidated.
Acknowledgement The authors declare that no competing interests exist. This work was supported by Grants from Youth Science Fund of Xinjiang Uighur Autonomous Region (2013211B50). The funding agency did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Reference Abdul Aziz, N.H., Kendall, D.A., and Pardon, M.C. 2012. Prenatal exposure to chronic mild stress increases corticosterone levels in the amniotic fluid and induces cognitive deficits in female offspring, improved by treatment with the antidepressant drug amitriptyline. Behavioural brain research 231, 29-39. Aguilera, G. 2011. HPA axis responsiveness to stress: implications for healthy aging. Experimental gerontology 46, 90-95. Aisa, B., Elizalde, N., Tordera, R., Lasheras, B., Del Rio, J., and Ramirez, M.J. 2009. Effects of 19
neonatal stress on markers of synaptic plasticity in the hippocampus: implications for spatial memory. Hippocampus 19, 1222-1231. Carvalho-Netto, E.F., Myers, B., Jones, K., Solomon, M.B., and Herman, J.P. 2011. Sex differences in synaptic plasticity in stress-responsive brain regions following chronic variable stress. Physiology & behavior 104, 242-247. de Quervain, D.J., Aerni, A., Schelling, G., and Roozendaal, B. 2009. Glucocorticoids and the regulation of memory in health and disease. Frontiers in neuroendocrinology 30, 358-370. Dole, N., Savitz, D.A., Hertz-Picciotto, I., Siega-Riz, A.M., McMahon, M.J., and Buekens, P. 2003. Maternal stress and preterm birth. American journal of epidemiology 157, 14-24. Feldt, K., Raikkonen, K., Pyhala, R., Jones, A., Phillips, D.I., Eriksson, J.G., Pesonen, A.K., Heinonen, K., Jarvenpaa, A.L., Strandberg, T.E., et al. 2011. Body size at birth and cardiovascular response to and recovery from mental stress in children. Journal of human hypertension 25, 231-240. Foster, C.J., Garber, J., and Durlak, J.A. 2008. Current and past maternal depression, maternal interaction behaviors, and children's externalizing and internalizing symptoms. Journal of abnormal child psychology 36, 527-537. Fumagalli, F., Franchi, C., Caffino, L., Racagni, G., Riva, M.A., and Cervo, L. 2009. Single session of cocaine intravenous self-administration shapes goal-oriented behaviours and up-regulates Arc mRNA levels in rat medial prefrontal cortex. The international journal of neuropsychopharmacology / official scientific journal of the Collegium Internationale Neuropsychopharmacologicum (CINP) 12, 423-429. Gale, C.R., and Martyn, C.N. 2004. Birth weight and later risk of depression in a national birth cohort. Br J Psychiatry 184, 28-33. Gronli, J., Bramham, C., Murison, R., Kanhema, T., Fiske, E., Bjorvatn, B., Ursin, R., and Portas, C.M. 2006. Chronic mild stress inhibits BDNF protein expression and CREB activation in the dentate gyrus but not in the hippocampus proper. Pharmacology, biochemistry, and behavior 85, 842-849. Hearing, M.C., Schochet, T.L., See, R.E., and McGinty, J.F. 2010. Context-driven cocaine-seeking in abstinent rats increases activity-regulated gene expression in the basolateral amygdala and dorsal hippocampus differentially following short and long periods of abstinence. Neuroscience 170, 570-579. Hsu, A.A., von Elten, K., Chan, D., Flynn, T., Walker, K., Barnhill, J., Naun, C., Pedersen, A.M., Ponaman, M., Fredericks, G.J., et al. 2012. Characterization of the cortisol stress response to sedation and anesthesia in children. The Journal of clinical endocrinology and metabolism 97, E1830-1835. Inberg, S., Elkobi, A., Edri, E., and Rosenblum, K. 2013. Taste familiarity is inversely correlated with Arc/Arg3.1 hemispheric lateralization. J Neurosci 33, 11734-11743. Johnson, B.N., and Yamamoto, B.K. 2010. Chronic stress enhances the corticosterone response and neurotoxicity to +3,4-methylenedioxymethamphetamine (MDMA): the role of ambient temperature. The Journal of pharmacology and experimental therapeutics 335, 180-189. Kajantie, E., Osmond, C., Barker, D.J., Forsen, T., Phillips, D.I., and Eriksson, J.G. 2005. Size at birth as a predictor of mortality in hood: a follow-up of 350 000 person-years. International journal of epidemiology 34, 655-663. Kapoor, A., Dunn, E., Kostaki, A., Andrews, M.H., and Matthews, S.G. 2006. Fetal programming of hypothalamo-pituitary-adrenal function: prenatal stress and glucocorticoids. The Journal of physiology 572, 31-44. Khalil, R., King, M.A., and Soliman, M.R. 2005. Testosterone reverses ethanol-induced deficit in spatial reference memory in castrated rats. Pharmacology 75, 87-92. Koenig, J.I., Kirkpatrick, B., and Lee, P. 2002. Glucocorticoid hormones and early brain development
20
in schizophrenia. Neuropsychopharmacology 27, 309-318. Kosten, T.A., Karanian, D.A., Yeh, J., Haile, C.N., Kim, J.J., Kehoe, P., and Bahr, B.A. 2007. Memory impairments and hippocampal modifications in
rats with neonatal isolation stress experience.
Neurobiology of learning and memory 88, 167-176. Kozisek, M.E., Middlemas, D., and Bylund, D.B. 2008. Brain-derived neurotrophic factor and its receptor tropomyosin-related kinase B in the mechanism of action of antidepressant therapies. Pharmacology & therapeutics 117, 30-51. Kramer, M.S., Lydon, J., Seguin, L., Goulet, L., Kahn, S.R., McNamara, H., Genest, J., Dassa, C., Chen, M.F., Sharma, S., et al. 2009. Stress pathways to spontaneous preterm birth: the role of stressors, psychological distress, and stress hormones. American journal of epidemiology 169, 1319-1326. Le Moal, M. 2007. Historical approach and evolution of the stress concept: a personal account. Psychoneuroendocrinology 32 Suppl 1, S3-9. Leung, E., Tasker, S.L., Atkinson, L., Vaillancourt, T., Schulkin, J., and Schmidt, L.A. 2010. Perceived maternal stress during pregnancy and its relation to infant stress reactivity at 2 days and 10 months of postnatal life. Clinical pediatrics 49, 158-165. Li, M., Hou, Y.Y., Lu, B., Chen, J., Chi, Z.Q., and Liu, J.G. 2009. Expression pattern of neural synaptic plasticity marker-Arc in different brain regions induced by conditioned drug withdrawal from acute morphine-dependent rats. Acta pharmacologica Sinica 30, 282-290. Li, M., Li, X., Zhang, X., Ren, J., Jiang, H., Wang, Y., Ma, Y., and Cheng, W. 2014. Effects of prenatal chronic mild stress exposure on hippocampal cell proliferation, expression of GSK-3alpha, beta and NR2B in
offspring during fear extinction in rats. Int J Dev Neurosci 35, 16-24.
Li, Z.Y., Zheng, X.Y., Gao, X.X., Zhou, Y.Z., Sun, H.F., Zhang, L.Z., Guo, X.Q., Du, G.H., and Qin, X.M. 2010. Study of plasma metabolic profiling and biomarkers of chronic unpredictable mild stress rats based on gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom 24, 3539-3546. Liu, X.H., Qian, L.J., Gong, J.B., Shen, J., Zhang, X.M., and Qian, X.H. 2004. Proteomic analysis of mitochondrial proteins in cardiomyocytes from chronic stressed rat. Proteomics 4, 3167-3176. Lobel, M., Cannella, D.L., Graham, J.E., DeVincent, C., Schneider, J., and Meyer, B.A. 2008. Pregnancy-specific stress, prenatal health behaviors, and birth outcomes. Health Psychol 27, 604-615. Maioli, S., Puerta, E., Merino-Serrais, P., Fusari, L., Gil-Bea, F., Rimondini, R., and Cedazo-Minguez, A. 2012. Combination of apolipoprotein E4 and high carbohydrate diet reduces hippocampal BDNF and arc levels and impairs memory in young mice. J Alzheimers Dis 32, 341-355. McLaughlin, K.J., Gomez, J.L., Baran, S.E., and Conrad, C.D. 2007. The effects of chronic stress on hippocampal morphology and function: an evaluation of chronic restraint paradigms. Brain Res 1161, 56-64. Moron, J.A., Gullapalli, S., Taylor, C., Gupta, A., Gomes, I., and Devi, L.A. 2010. Modulation of opiate-related signaling molecules in morphine-dependent conditioned behavior: conditioned place preference to morphine induces CREB phosphorylation. Neuropsychopharmacology 35, 955-966. Obel, C., Hedegaard, M., Henriksen, T.B., Secher, N.J., Olsen, J., and Levine, S. 2005. Stress and salivary cortisol during pregnancy. Psychoneuroendocrinology 30, 647-656. Prut, L., and Belzung, C. 2003. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. European journal of pharmacology 463, 3-33. Rizk, P., Salazar, J., Raisman-Vozari, R., Marien, M., Ruberg, M., Colpaert, F., and Debeir, T. 2006. The alpha2-adrenoceptor antagonist dexefaroxan enhances hippocampal neurogenesis by increasing the survival and differentiation of new granule cells. Neuropsychopharmacology 31, 1146-1157.
21
Salomon, S., Bejar, C., Schorer-Apelbaum, D., and Weinstock, M. 2011. Corticosterone mediates some but not other behavioural changes induced by prenatal stress in rats. Journal of neuroendocrinology 23, 118-128. Shepherd, J.D., and Bear, M.F. 2011. New views of Arc, a master regulator of synaptic plasticity. Nature neuroscience 14, 279-284. Simon, P., Dupuis, R., and Costentin, J. 1994. Thigmotaxis as an index of anxiety in mice. Influence of dopaminergic transmissions. Behavioural brain research 61, 59-64. Sirianni, R.W., Olausson, P., Chiu, A.S., Taylor, J.R., and Saltzman, W.M. 2010. The behavioral and biochemical effects of BDNF containing polymers implanted in the hippocampus of rats. Brain Res 1321, 40-50. Steward, O., and Worley, P. 2002. Local synthesis of proteins at synaptic sites on dendrites: role in synaptic plasticity and memory consolidation? Neurobiology of learning and memory 78, 508-527. Waddell, S., and Jenkins, J.R. 1995. arg3+, a new selection marker system for Schizosaccharomyces pombe: application of ura4+ as a removable integration marker. Nucleic acids research 23, 1836-1837. Wadhwa, P.D. 2005. Psychoneuroendocrine processes in human pregnancy influence fetal development and health. Psychoneuroendocrinology 30, 724-743. Weinstock, M. 2005. The potential influence of maternal stress hormones on development and mental health of the offspring. Brain, behavior, and immunity 19, 296-308. Willner, P. 1997. Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology 134, 319-329. Yang, J., Pei, Y., Pan, Y.L., Jia, J., Shi, C., Yu, Y., Deng, J.H., Li, B., Gong, X.L., Wang, X., et al. 2013. Enhanced antidepressant-like effects of electroacupuncture combined with citalopram in a rat model of depression. Evid Based Complement Alternat Med 2013, 107380. Yu, I.T., Lee, S.H., Lee, Y.S., and Son, H. 2004. Differential effects of corticosterone and dexamethasone on hippocampal neurogenesis in vitro. Biochemical and biophysical research communications 317, 484-490. Zhong, N., Scearce-Levie, K., Ramaswamy, G., and Weisgraber, K.H. 2008. Apolipoprotein E4 domain interaction: synaptic and cognitive deficits in mice. Alzheimers Dement 4, 179-192.
22
Figure Captions Figure 1 The comparison of plasma corticosterone level between PS and PC group The figure shows that changes in plasma corticosterone level of PS were noted after stress model is established successfully. Data were analyzed using repeated measurement analysis of variance. Each column represents mean ± SD. Number of animals in each group = 10. *P < 0.05 vs. PC group.
Figure 2 The comparison of behaviors in open-field test between PS and PC groups At regular time intervals throughout the 21 days of stress, differences in horizontal movement (A) and vertical movement (B) were observed between CUMS and control groups. Data were analyzed using repeated measurement analysis of variance. Each column represents mean ± SD. Number of animals in each group=10. *P < 0.05 vs. PC group.
23
Figure 3 The comparison on behaviors of liquid consumption test between PS and PC group At regular time intervals throughout the 21 days of stress, differences in pure water consumption (A), sugar water consumption (B), total liquid consumption (C) and sucrose preference (D) were detected between PS and PC groups. Data were analyzed using repeated measurement analysis of variance. Each column represents mean ± SD. Number of animals in each group = 10. *P < 0.05 vs. PC group.
24
Figure 4 The comparison on the body weight between PS and PC groups At regular time intervals throughout the 21 days of stress, differences in maternal body weight were detected between PS and PC groups. The growth rate of maternal body weight in PS was lower than PC group. Data were analyzed using repeated measurement analysis of variance. Number of animals in each group = 10. Each column represents mean ± SD. *P < 0.05 vs. PC group.
25
Figure 5 The comparison on the basic situation of reproduction between PS and PC group At regular time intervals throughout the 21 days of stress in pregnancy, observations for differences in the number of offspring (A), days of pregnancy (B) and the proportion of male and female (C) were made between PS and PC groups. The number of offspring and days of pregnancy in PS group was less than the PC group. Data were analyzed using student’s t test. Each column represents mean ± SD. Number of animals in each group = 10. *P < 0.05 vs. PC group.
26
Figure 6 The comparison between PS offspring and PC offspring in body weight and plasma corticosterone level Differences in body weight and plasma corticosterone level were detected between PS offspring and PC offspring. The growth rate of offspring in PS was lower than PC group. The plasma corticosterone level of PS offspring was higher than that of PC offspring. On different postnatal days, data were respectively analyzed using student’s t test. Each column represents mean ± SD. Number of animals in each offspring group = 16 (50% male, 50% female) *P < 0.05 vs. PC offspring.
27
Figure 7 Comparison in escapes latency and crossing platform between PS and PC offspring groups Differences in escapes latency and crossing platform were detected between PS and PC offspring from PND 42. The rate of increase of escapes latency and crossing platform in PS group was lower than PC group. Data were analyzed using repeated measurement analysis of variance. Each column represents mean ± SD. Number of animals in each offspring group = 16 (50% male, 50% female) *P < 0.05 vs. PC offspring.
28
Figure 8 Histological section of hippocampus Representative photomicrographs of the hippocampal CA1 area (×200, A), hippocampal neurons (×1 0000, B) and Hippocampal synaptic cell (×5 0000, C)
Figure 9 The effects of maternal stress during pregnancy on the BDNF and Arc expression in the offspring’s hippocampus Differences in BDNF and Arc 29
expression in the hippocampus were detected between PS offspring and PC offspring. The BDNF mRNA, protein and Arc protein expression of PS offspring were lower than that of PC offspring. At different postnatal day, data were respectively analyzed using student’s t test. Each column represents mean ± SD. Number of animals in each offspring group = 16 (male, female in half). *P < 0.05 vs. PC offspring.
30
Table 1 Correlations between learning and memory of offspring and the determination results of their mothers PS PS offspring Plasma corticosterone
Horizontal movement
Sucrose preference
Escape latency
0.556*
-0.464*
-0.626*
Crossing platform
-0.441*
0.676*
0.630*
* P<0.05 Table 2 Correlations between learning and memory and protein expression of offspring Parameters
BDNF
Arc
Escape latency
-0.589*
-0.421*
Crossing platform
0.556*
0.458*
* P<0.05
31