Maternal infection during late pregnancy increases anxiety- and depression-like behaviors with increasing age in male offspring

Brain Research Bulletin 87 (2012) 295–302

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Maternal infection during late pregnancy increases anxiety- and depression-like behaviors with increasing age in male offspring Mohsen Enayati a , Jalal Solati b , Mohammad-Hassan Hosseini c , Hamid-Reza Shahi a , Golshid Saki d , Ali-Akbar Salari e,∗ a

Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran Department of Biology, Karaj Branch, Islamic Azad University, Karaj, Iran c Department of Veterinary Medicine, Karaj Branch, Islamic Azad University, Karaj, Iran d Department of Biology, Science and Research Branch, Islamic Azad University, Fars, Iran e Department of Cellular and Molecular Biology, School of Science, Tehran North Branch, Islamic Azad University, P.O. Box. 19585-936, Tehran, Iran b

a r t i c l e

i n f o

Article history: Received 2 June 2011 Received in revised form 8 August 2011 Accepted 22 August 2011 Available online 27 August 2011 Keywords: Lipopolysaccharide Maternal infection Late pregnancy Anxiety Depression Cytokine Corticosterone Mice

a b s t r a c t Scientific reports suggest that the exposure to long-term stressors throughout or during late gestation increase anxiety- and depression-like behaviors of offspring in their later life. Moreover, several studies concluded that increasing age correlates with increased anxiety behaviors in humans and rodents. In the present study, we assessed the effects of prenatally administration of equal lipopolysaccharide (LPS) doses in various points of late gestation (days 15, 16, and 17) period, on neuroendocrine and immunological responses of pregnant mice, and subsequent long-lasting consequences of anxiety and depression with increasing age in male offspring at postnatal days (PD) 40 and 80. Four hours after the LPS injection, levels of corticosterone (COR) and pro-inflammatory cytokines (PIC) in pregnant mice, as compared to the control dams, were increased significantly. Furthermore, maternal inflammation raised the levels of COR, anxiety- and depression-like behaviors with increasing age in male offspring in comparison with saline male offspring. These data support other studies demonstrating that maternal stress increases the levels of anxiety and depression in offspring. Additionally, our data confirm other findings indicating that increasing age correlates with increased anxiety or depression behaviors in humans and rodents. Findings of this study suggest that time course of an inflammation response or stressor application during various stages of gestation and ages of offspring are important factors for assessing neuropsychiatric disorders. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Findings of the experimental and epidemiological studies indicated that the major psychiatric disorders, like anxiety and depression can influence any aspects of life. Today, neuroscience researchers believe that the origins and causes of anxiety- and depression-like behaviors could be rooted in multifactorial dysfunctions including both environmental and genetic disorders [5]. Moreover, anxiety is often associated with depression [12] and genes likely have a crucial role in the etiology and correlation of these two behaviors [22,39,77]. For many years, many scientists put great effort into seeking out and understanding important genetic factors involved in these behaviors of humans. During recent years, considerable insights have been gained into the effects of prenatal infection or stress on the development of fetal in animals and humans [7,14,29,47,61], but unfortunately the factors

∗ Corresponding author. Tel.: +98 919 4099673; fax: +98 21 22977853. E-mail address: [email protected] (A.-A. Salari). 0361-9230/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2011.08.015

mediating these effects on neuropsychiatric disorders including anxiety, depression, schizophrenia and autism are not still clear. Lipopolysaccharide (LPS, bacterial endotoxin) administration is a well-established and widely accepted mouse model of maternal infection [14]. According to our recent study, systemic LPS administration in pregnant mice leads to a higher production of pro-inflammatory cytokines (PIC) such as interleukin (IL)-6, tumor necrosis factor-alpha (TNF-␣) and IL-1␤, corticostrone (COR) secretion, and hypothalamic–pituitary–adrenal (HPA) axis activation [7]. Systemic exposure to LPS resulted in a strong but time-limited inflammatory cytokine response in the host. Cytokines play an important role in normal brain development [78], synaptic plasticity, neurogenesis and neuromodulation [40,45], and are involved in the death and dysfunction of neurons following an injury in the adult brain [2] and can be considered as a mediating agent for linking maternal inflammation and neurodevelopmental damage. Furthermore, glucocorticoids are crucial agents for normal brain development [33,44,53], neuronal survival [66], differentiation [60] and both structural and functional development of synapses [6,36]. Actually these agents have profound impacts on the brain, because

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any fluctuations and disturbances in their circulating levels and patterns can impose harmful effects. Therefore, the presence of these elements imposes multiple risk factors for development of fetus during different stages of pregnancy. Previous studies indicated that the exposure time points of prenatal immune challenge, postnatal age of the offspring, and the dose of immunogens are key factors in induction of different neurophysiological, behavioral and immunological responses in offspring [7,9,10,70]. Scientific reports showed that exposure to long-term stress throughout or during late gestation increased anxiety- and depression-like behaviors of offspring in their later life [3,4,21,24,26,51,71]. Moreover, several studies concluded that increasing age correlates with an increased level of anxiety behaviors in humans and rodents [27,37,64,65]. However, so far little attention has been devoted to comparison of gestation length and duration of exposure to stress in this period between humans and laboratory animals. The gestation period for mouse and human are 20 days and 40 weeks, respectively. Therefore, each gestation day of a mouse can be equivalent to a 2-week gestation period of a human. Given the importance of this analogy, it can be concluded that duration or severity of exposure to stress or infection during pregnancy and time course of an inflammation or stress response are important for determining the nature of extracted effects on the stress responses of offspring in their later life stages. Therefore, the aim of the present study was to investigate the effects of time-limited inflammation response in days 15, 16 and 17 of late gestation on the development of anxiety- and depressionlike behaviors in adolescent and adult male offspring. Gestational days (GD) 15, 16 and 17 were chosen as the time points for assessment [8,23,30,34] and then the relation between prenatal infection and development of neuropsychiatric disorders in later life was evaluated. To this end we assessed the effects of prenatally administration of equal LPS doses at the aforementioned time points on the concentrations of PIC and COR in pregnant mice and levels of COR, anxiety- and depression-like behaviors in the elevated plus maze (EPM), and forced swimming test (FST) in their male offspring at postnatal days (PD) 40 and 80. All of these parameters were separately studied in adolescent and adult male offspring.

2.2. Induction of maternal infection In this study the pregnant mothers were randomly divided into 3 clusters, each of which consisted of four main groups: maternal control group (in which pregnant mice were subjected to the same treatment conditions, but injected with saline), and three maternal LPS groups (received different LPS doses: 50, 300, and 500 ␮g/kg). The above doses of LPS were chosen based on other previous studies on pregnant rodents investigating the necessary conditions for induction of inflammatory cytokines [14]. In order to model a physiological maternal infection in the late gestation, at GD 15, 16 and 17, LPS (Salmonella enterica serotype entritidis, Sigma Co., USA) was dissolved in sterile pyrogen-free saline (0.9% NaCl) and then was administered intraperitoneally (i.p.) to pregnant mice. Injections were performed between 10:00 and 11:00 A.M. Each injection was performed with a 1-ml insulin syringe needle preloaded with 50 ␮l saline or LPS solution. 2.3. Study design Total of 288 pregnant mice were divided into the 12 main experimental groups (as mentioned above, 3 clusters, each consists of 4 groups) and each main group consisted of 24 pregnant dams, some of which were used for measuring the PIC and COR levels and the remaining animals were allowed to give birth and nurture their offspring normally (12 pregnant mice were used for each variable evaluation). All of contraindication agents to the experimental procedures were excluded, including any maternal interventions after LPS exposure such as changing the cage for cleaning during late gestation and neonatal period, prenatal and postnatal weighing of mother and pups, any type of invasive interventions which may modify parental or maternal care which could result in any variations in the neurophysiological and behavioral traits of offspring. The prenatal LPS treatment did not affect the pregnancy length (19–20 day), and we did not find any significant difference in litter size between prenatal LPSand saline-treated groups (litter size: 10–12). Two separate experiments were conducted. The anxiety- and depression-like behaviors of adolescent (PD 40) and adult (PD 80) male offspring were assessed the EPM and FST. To avoid litter effect, no more than one mouse per dam was used for each of the above experiments (12 offspring were included in each group per 12 mothers; ratio of one to one). Following the experiment of the EPM assessment, COR serum levels in male offspring were measured. In all experiments, each male offspring was tested once. 2.4. Blood sampling and testing Cardiac puncture blood collection was performed on the pregnant dams and the male offspring. Animals were anesthetized by an i.p. injection of ketamine hydrochloride (50 mg/kg; Alfasan, Woerden-Holland) plus Xylazine (5 mg/kg; Alfasan, Woerden-Holland). The entire sampling procedure was completed within 8 min of removing the animal from its living cage, which is rapid enough to perform a reliable assessment of COR and PIC at the time of sampling, without any undesirable effect of disturbance or anesthesia. Their trunk blood was collected into sterile tubes and allowed to clot on ice for a minimum of 30 min before centrifugation at 3000 rpm for 15 min, serum was collected into sterile vials and stored at −20 ◦ C and −80 ◦ C until assayed for the COR and PIC contents, respectively.

2. Materials and methods 2.5. Enzyme-linked immunosorbent assay 2.1. Breeding and animals Ten to 11-week-old male and female NMRI mice were obtained from the animal house of Pasteur Institute of Iran (Tehran, Iran), and breeding began after a 2-week period of acclimatization to the new animal holding room. Animals were housed in standard polycarbonate cages (4 per cage) in a room with a 12:12 h light/dark cycle (lights on 07:00–19:00 h), controlled temperature (23 ± 1 ◦ C) and free access to food and water. These conditions were kept as a standard housing condition in all stages of experiments. In order to induction of the estrus cycle in female mice, two male and four female mice were put in a partitioned cage with no physical contact. In this condition the female mice were exposed to the male pheromones for 3 days to induce and synchronize the estrus cycle in females. In order to facilitate of mating, male and female mice were kept together one-by-one in a cage. Successful mating was confirmed next morning (7:00 A.M.) with the presence of vaginal plug, and that day was referred to as GD 0 [46]. Once a pregnant female was identified, it was removed from the breeding cage and housed individually in a standard cage. Following delivery, in order to avoid any potential alterations in maternal behavior directly related to culling, litters were left untouched with their mother. On the day 21, litters were weaned by removal of the mother and then were housed with same sex litter-mates (4 animals per cage). Only the male pups were enrolled in the experiments. Animals were housed in a standard housing condition. The cages were changed twice a week, except during the late pregnancy and neonatal period. All behavioral tests were performed during the light period (between 13:00 and 17:00 h) under a dim light illumination and all experiments were performed according to the protocols approved by Institutional Animal Ethics Committee of Islamic Azad University (IAU-IAEC) and were conducted under the recommended conditions of the Guide for the Care and Use of Laboratory Animals of the National Institute of Health (NIH).

Circulating levels of COR (Immunodiagnostic systems Company, AC-14F1, UK), IL-1␤ (Immuno-Biological Laboratories, IB49700, USA), IL-6 (BioSource International, CA 93012, USA), and TNF-␣ (U-Cytech, CT 302 Netherlands) in maternal, adolescent and adult male offspring serum were evaluated using the Sandwich Enzyme-linked Immunosorbent Assay (ELISA) according to the manufacturer’s instructions. All samples and standards were assayed in duplicate. 2.6. Behavioral tests equipment and procedures 2.6.1. Anxiety test The elevated plus-maze, which is one of the well-known methods for testing of anxiety, was used in the present study. The EPM was a plus-shaped apparatus, constructed from wood, elevated to a height of 50 cm above the floor. This apparatus was consisted of a central platform (5 cm × 5 cm), two open arms (30 cm × 5 cm), and two equal closed (30 cm × 5 cm × 15 cm) arms opposite to each other with an open roof [42]. The EPM was placed in the center of a quiet and dimly lit room. The animal behavior was directly observed using a mirror, suspended at an angle above the EPM. Behavioral data were collected by a blind observer who used a chronometer and quietly sat at distance of a 1 m behind one of the closed arms of the maze. Mice were placed individually in the center of the EPM, facing one of the open arms. A relatively dark box was used to hold the mice in before placing on the maze in order to increase their exploratory behavior [56]. The observer measured: (1) time spent in the open arms, (2) time spent in the closed arms, (3) number of entries into the open arms, and (4) number of entries into the closed arms during the 5-min test period. A mouse was considered to be on the open or closed arms (spent time) whenever all four paws were in the respective arm. An entry was defined as all four paws on the arm. The EPM was cleaned with distilled water between each test.

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Fig. 1. Serum levels of IL-1␤ (A), IL-6 (B), TNF-␣ (C) and COR (D) in the pregnant dams exposed to the saline or equal LPS doses on GDs 15, 16 and 17. Serum PIC and COR levels were measured 4 h after administration of drug or vehicle in pregnant mice and more details of the results of analysis are provided in Section 3. The data are presented as mean ± S.E.M. (N = 12). Significant differences: *P < 0.05, **P < 0.01 and ***P < 0.001, compared to saline-administered dams.

For the purpose of analysis [20,54,55], percent of open-arm time [OAT % (time in open arm/time in open + closed arm) × 100], and percent of open arm entries [OAE % (number of open arm entries/number of open arm + closed arm entries) × 100] were defined and employed as measures of anxiety. The total number of open arms entries, as well as the total number of closed arm entries was defined as an index of general locomotor activity (LMA) [59,68]. 2.6.2. Depression test The forced swimming test remains one of the most widely used tools for assessing antidepressant activity. To describe this behavioral model in mice, the following procedure was adopted, mice were individually placed into the transparent glass cylinders (Height: 25 cm, Diameter: 10 cm), filled with water to a height of 15 cm and maintained at 25 ± 1◦ C. The water was replaced by fresh water between each test. The total duration of immobility was recorded during the last 4 min of the 6 min testing period. Each mouse was judged to be immobile when it ceased struggling and remained floating motionless in the water and making only those movements necessary to keep its head above water. A decrease in the duration of immobility is an indicative of an antidepressant-like effect [15,32,57,58]. 2.7. Statistical analysis The statistical analyses were performed using the “Statistical Package for Social Sciences” (SPSS, Version 18). All data on pregnant mothers and their offspring in each GD were separately analyzed using one-way ANOVA followed by Tukey’s HSD test as post hoc multiple comparisons. Three-way ANOVA was used for the evaluation of the interactions between all factors (age × GD × treatment). Data are presented as the mean ± standard error of the mean (S.E.M.). The P value of less than 0.05 (P < 0.05) was considered statistically significant.

3. Results 3.1. Gestational cytokines and corticosterone Concentration of IL-1␤ (Fig. 1A), IL-6 (Fig. 1B), TNF-␣ (Fig. 1C) and COR (Fig. 1D) were assayed in maternal serum, 4 h after the administration of LPS or saline. Minimal serum levels of these cytokines were observed in saline-treated dams. As it was expected, LPS administration resulted in induction of all three cytokines in

maternal serum. In the late gestation, after administration of LPS on GD 15, significant increase was observed in the concentration of the serums IL-1␤ (300 and 500 ␮g/kg) [F(3, 44) = 4.52, P < 0.009] and IL-6 (500 ␮g/kg) [F(3, 44) = 4.86, P < 0.006] in mothers. On GD 16, LPS at the doses of 300 and 500 ␮g/kg resulted in significant increases in the levels of the serums IL-1␤ [F(3, 44) = 4.03, P < 0.02] and IL-6 [F(3, 44) = 11.53, P < 0.001] in dams, however, on GD 17, all three doses of LPS led to significant increases in the levels of these two cytokines (IL-1␤: [F(3, 44) = 4.88, P < 0.006]; IL-6: [F(3, 44) = 8.26, P < 0.001]). Furthermore, equal LPS doses on the GDs 15 [F(3, 44) = 359.73, P < 0.001], 16 [F(3, 44) = 360.53, P < 0.001] and 17 [F(3, 44) = 474.73, P < 0.001] significantly increased the levels of TNF-␣, as compared with the serum of saline dams. In addition, maternal serum levels of COR, as compared with the saline mothers, were significantly elevated in the LPS-treated dams on the GD 15 [F(3, 44) = 3.42, P < 0.03] and GDs 16 [F(3, 44) = 11.19, P < 0.001] and 17 [F(3, 44) = 13.57, P < 0.001] at the doses of 500 ␮g/kg, and 300 and 500 ␮g/kg, respectively. 3.2. Adolescent and adult offspring anxiety Previous studies suggested that increasing age correlates with an increased level of anxiety behaviors in rodents. First, we tested this hypothesis by exploring whether maternal inflammation during different stages of late gestation period can influence the anxiety-related behaviors with increasing age in adolescent and adult male offspring. Anxiety was assessed using the EPM technique. In the adolescent male pups (Fig. 2), one-way ANOVA revealed that the maternal LPS exposure on the GDs 16 (OAT %: [F(3, 44) = 3.40, P < 0.03]; OAE %: [F(3, 44) = 4.56, P < 0.008]) and 17 (OAT %: [F(3, 44) = 3.65, P < 0.03]; OAE %: [F(3, 44) = 3.87, P < 0.02]), decreased the OAT % and OAE % significantly, indicating high levels of anxiety in the LPS adolescent pups on PD 40. No significant change in the above variables was observed for the GD 15 (OAT

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Fig. 2. Effects of the maternal exposure to saline or equal LPS doses during the different stages of late gestation on anxiety-related behavior of the adolescent male offspring (PD 40) in the EPM. More details of the analyses results are provided in Section 3. Each bar represents mean ± S.E.M. (N = 12) of the open arm time % (A), open arm entries % (B) or locomotor activity (C). Significant differences: *P < 0.05, compared to the adolescent saline male offspring.

%: [F(3, 44) = 0.51, P > 0.05]; OAE %: [F(3, 44) = 0.19, P > 0.05]). Also, no significant change was observed in the locomotor activity (GD 15: [F(3, 44) = 1.08, P > 0.05]; GD 16: [F(3, 44) = 0.36, P > 0.05]; GD 17: [F(3, 44) = 1.31, P > 0.05]) following the prenatal LPS exposure, as compared to the saline adolescent male pups. Post hoc analysis showed that the significant effects were obtained by the doses of 300 and 500 ␮g/kg on GD 16, and the dose of 500 ␮g/kg on the GD 17, while the administration of the all three LPS doses (50, 300, and 500 ␮g/kg) on GD 15, the dose 50 ␮g/kg on GD 16, and the doses 50 and 300 ␮g/kg on the GD 17 showed no significant effects on anxiety behavior of adolescent male offspring in the EPM. One-way ANOVA indicated that in the adult male offspring (Fig. 3), the prenatal LPS exposure on the GDs 15 (OAT %: [F(3, 44) = 4.09, P < 0.02]; OAE %: [F(3, 44) = 6.99, P < 0.002]), 16 (OAT %: [F(3, 44) = 6.46, P < 0.002]; OAE %: [F(3, 44) = 9.38, P < 0.001]) and 17

Fig. 3. Effects of the maternal exposure to saline or the equal LPS doses during different stages of late pregnancy on anxiety-related behavior of adult male offspring (PD 80) in the EPM. More details of the statistical analyses results and post hoc comparisons are provided in Section 3. Each bar represents mean ± S.E.M. (N = 12) of the open arm time % (A), open arm entries % (B) or locomotor activity (C). Significant differences: *P < 0.05, **P < 0.01 and ***P < 0.001, compared to the saline adult male offspring.

(OAT %: [F(3, 44) = 7.25, P < 0.001]; OAE %: [F(3, 44) = 5.10, P < 0.005]) decreased the OAT % and OAE % significantly; indicating higher levels of anxiety in the LPS adult male offspring on the PD 80. No significant change was observed in the locomotor activity (GD 15: [F(3, 44) = 0.72, P > 0.05]; GD 16: [F(3, 44) = 1.23, P > 0.05]; GD 17: [F(3, 44) = 3.24, P > 0.03]) following the maternal LPS exposure, as compared to the saline adult male offspring. Post hoc analysis showed that the significant impacts were obtained by the 300 and 500 ␮g/kg doses on the GDs 15 and 16, and by all doses on the GD 17, while the administration of 50 ␮g/kg dose on the GDs 15 and 16 had no significant effects on anxiety behavior of adult male offspring in the EPM test. Finally, three-way analyses revealed overall main effects of age (OAT% [F(1, 264) = 120.62, P < 0.001], OAE % [F(1, 264) = 54.29,

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Fig. 4. Serum COR levels in the saline or LPS adolescent (PD 40) and adult (PD 80) male offspring after exposure to EPM were measured. Each bar represents mean ± S.E.M. (N = 12) of COR levels. Significant differences: *P < 0.05 and **P < 0.01, compared to the saline adolescent and adult male offspring.

P < 0.001] and LMA [F(1, 264) = 16.58, P < 0.001]), GD (OAT% [F(2, = 11.40, P < 0.001] and OAE % [F(2, 264) = 10.45, P < 0.001]) and treatment (OAT% [F(3, 264) = 16.24, P < 0.001] and OAE % [F(3, 264) = 16.06, P < 0.001]). Significant interactions existed between age × GD in OAT% [F(2, 264) = 11.40, P < 0.001] and OAE% [F(2, 264) = 10.17, P < 0.001], GD × treatment in OAE % [F(6, 264) = 2.80, P < 0.02], but were no significant differences between the interactions of age × treatment and age × GD × treatment in OAT% and OAE% or LMA in offspring. These results demonstrate that maternal exposure to the equal LPS doses in different stages of late gestation raised the levels of anxiety with increasing age in male offspring, which are not previously reported in other similar previous studies.

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Fig. 5. Effects of the maternal exposure to saline or equal LPS doses during the different stages of late gestation on depression-related behavior of adolescent (PD 40) and adult (PD 80) male offspring in the FST test. More details of the results of statistical analysis and post hoc comparisons are provided in Section 3. Each bar represents mean ± S.E.M. (N = 12) of total duration of immobility. Significant differences: *P < 0.05 and **P < 0.01, compared to the saline adolescent and adult male offspring.

264)

to EPM (Fig. 4). Moreover, three-way analyses revealed overall main effects of age [F(1, 264) = 30.38, P < 0.001], GD [F(2, 264) = 7.37, P < 0.002] and treatment [F(3, 264) = 9.04, P < 0.001]. Significant interactions existed between age × GD [F(2, 264) = 12.22, P < 0.001], age × treatment [F(3, 264) = 5.60, P < 0.002], and GD × treatment [F(6, 264) = 2.40, P < 0.03]. No significant difference was observed between age × GD × treatments [F(6, 264) = 0.82, P > 0.05] among offspring. Our data illustrated that prenatal exposure to the equal LPS doses in different stages of late gestation raised the levels of COR with increasing age in male offspring (Fig. 4).

3.3. Adolescent and adult offspring corticosterone

3.4. Adolescent and adult offspring depression

Following the exposure to the EPM, on the PDs 40 and 80, the concentration of COR was assayed in the male offspring serum (Fig. 4). For all three doses, there were no significant changes in the levels of COR of the LPS adolescent male pups serum in comparison with the serum of saline pups on GDs 15 [F(3, 44) = 0.70, P > 0.05], 16 [F(3, 44) = 1.11, P > 0.05] and 17 [F(3, 44) = 3.23, P < 0.04]. However, on GD 17, a significant change was observed for the adolescent male offspring born from the dams exposed to 500 ␮g/kg dose. Furthermore, one-way ANOVA showed a significant increase in the concentration of COR in the adult male offspring serum born from the dams treated with 500 ␮g/kg dose at GD 15 [F(3, 44) = 3.90, P < 0.02], 300 and 500 ␮g/kg on GDs 16 [F(3, 44) = 4.18, P < 0.02] and 17 [F(3, 44) = 7.13, P < 0.002], in comparison with the saline adult offspring serum after exposure

First, we explored the long-term effects of maternal exposure to equal doses of LPS or saline during different stages of late gestation on depression-related behaviors with increasing age in the adolescent and adult male offspring in the FST (Fig. 5). In adolescent male pups, one-way ANOVA showed that maternal LPS exposure on GD 17 [F(3, 44) = 5.86, P < 0.003] increased the total duration of immobility at the dose of 500 ␮g/kg, indicating high levels of depression behaviors in the LPS adolescent pups on PD 40, while injection of equal LPS doses on GDs 15 [F(3, 44) = 1.51, P > 0.05], 16 [F(3, 44) = 2.06, P > 0.05], and 50 and 300 ␮g/kg at GD 17 did not show any significant change in the LPS adolescent male, in comparison with the saline adolescent pups. On the other hand, Post hoc analysis showed that administration of the 300 ␮g/kg dose on GD 16[F(3, 44) = 6.25, P < 0.002], and doses of 300 and 500 ␮g/kg on GD 17 [F(3, 44) = 7.68,

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P < 0.001] resulted in the significant effects in adult male offspring. However, one-way ANOVA showed that LPS administration at all three doses on GD 15 [F(3, 44) = 1.54, P > 0.05], two doses of 50 and 500 ␮g/kg on GD 16, and also the dose of 50 ␮g/kg on GD 17 in adult male offspring did not resulted in any significant effects, as compared to the saline adult male offspring. Furthermore, three-way analyses revealed overall main effects of age [F(1, 264) = 4.77, P < 0.03], GD [F(2, 264) = 7.61, P < 0.002] and treatment [F(3, 264) = 9.05, P < 0.001]. Significant interactions existed between age × GD [F(2, 264) = 46.95, P < 0.001], age × treatment [F(3, 264) = 3.89, P < 0.02], and GD × treatment [F(6, 264) = 3.73, P < 0.002]. No significant difference was observed between the interactions of age × GD × treatments [F(6, 264) = 1.70, P > 0.05] among offspring. Using these data, it can be concluded that maternal exposure to the equal doses of LPS in different stages of late gestation elevated the levels of depression with increasing age in male offspring, which were not previously reported in similar previous studies.

4. Discussion In the present study, our primary results indicate that systemic LPS challenges in first 3 days of late gestation influence neurophysiological and immunological responses of pregnant mice by induction of PIC, COR and activation of the HPA axis, especially in higher LPS doses. Thus, these changes could act directly or indirectly via regulation of fetal gene expression and alterations in placental metabolism on the fetal development. Our data showed that maternal LPS exposure in different stages of late gestation increased anxiety, COR levels and depression with increasing age in male offspring. A further interesting observation of the current study was that female offspring, as compared to male offspring, showed an increased level of severity of anxietyand depression with increasing age (Salari et al., unpublished data). However, these results are consistent with the other studies showing that exposure to stress throughout or during late gestation correlates with increased anxiety and depressant behaviors in rodents’ offspring. For instance, (I) several studies showed the higher levels of anxiety in stressed male and female rats offspring either throughout gestation (PD 60; PD 180) [21,24], or during some periods, for example, application of stress agent three times daily from day 17 to 21 (PD 120) [51,71], and maternal LPS exposure on gestational day 17 in the EPM (PD 240) [34]; (II) prenatal stress throughout gestation or during late pregnancy in mice and rats increases the duration of the immobility period in the FST, and this increase is more evident in the females than males (PD 80 and 200; PD 55) [3,4,26]. To be sure, our findings are similar to the data reported on the human studies showing that the onset of pathological anxiety, which often manifests at a young age, persists throughout adolescent and continues into adulthood [16]. These gestational days (15, 16 and 17) are corresponding to the neurogenesis of hippocampus and nucleus accumbens in mice (http://www.translatingtime.net), which plays the crucial role in the modulation of anxiety and depression in humans and rodent [13,17,49,68]. Several findings from previous studies in rodents show alteration of the function of the HPA axis, molecular and neurotransmitter systems’ changes in brains of offspring after maternal inflammation or stress [14,29,38]. It has long been known that placenta plays a pivotal role in the regulation of maternal factors transfer to the fetus [52]. During recent years, some authors have reported that IL-6 and glucocorticoids during pregnancy can move across the placenta and mediate the brain damage and subsequent neurodevelopmental disorders in offspring [19,33,44,53,63,67]. As described in the results, we found a significant link between the increase in the levels of maternal IL-6 and COR, and neurobehavioral changes, including increased anxiety and depression as well

as HPA axis hyperactivity correlate with increasing age in male offspring. Therefore, we believe that maternal IL-6 and glucocorticoids are the main candidates for the mediation of developmental alterations in offspring. In line with this hypothesis, a recently published study, conducted by Hsiao and Patterson, demonstrates a relationship between activation of the maternal immune system and endocrine changes in the placenta via IL-6, which could alter fetal development through an indirect mechanism [35]. Moreover, previous studies indicated that prenatal exposure to cortisol or dexamethasone has long-term neurodevelopmental consequences on fetus and offspring in humans and rodents [11,41]. In this respect, human studies show that increased cortisol exposure affects the expression of over a thousand genes in fetal brain cells and this exposure affect the function of the placenta, including the expression and activity of 11beta-hydroxysteroid dehydrogenase 2 (11␤-HSD2), the main barrier to the placental passage of glucocorticoids [28,43,62,76]. Many studies demonstrate that in rodents, prenatal corticosteroid treatments have multiple effects on hippocampal cell proliferation, neurotransmitter turnover, and receptor expression [11,48,69]. On the other hand, maternal dexamethasone raises corticosterone and adrenocorticotropic hormone (ACTH) levels in the adult offspring [41,50,75]. It seems these effects demonstrate an alteration in the feedback of the HPA axis at the level of the hypothalamus, because the levels of corticotropin-releasing hormone (CRH) mRNA were elevated in the paraventricular nucleus whereas, the levels of hippocampal mineralocorticoid (MR) and glucocorticoid (GR) were both decreased [18,74]. Hippocampus is a part of the brain that is important both for anxiety and depression, and the control of the HPA axis. This could explain why the COR response to a new stressor is both increased and prolonged; there is less feedback inhibition via COR acting on its receptors. Furthermore, a positive correlation between anxiety- and depression-like behaviors and increasing age was observed in the mice model of prenatal infection. Age span of the animal ranged from PD 40 to PD 80, which contains sexual maturation in male mice with an increase in androgen production. An interesting question is about the role of androgens as an intervening agent in the increase of anxiety and depression levels with increasing age in prenatal infection model of male offspring. It was shown that there was a negative correlation between testosterone level and anxiety level in male rodents [1,25] in which the more the level of this hormone the lower the anxiety level was observed. Moreover, this negative association was reported in adolescent human males and females demonstrating high levels of anxiety and depression, behavioral disturbance, and attention problems, caused by low levels of testosterone [31]. Previous studies showed that males with a history of prenatal stress show lower levels of plasma testosterone [73] and also lower activity of beta-hydroxysteroid dehydrogenase, an enzyme responsible for the final conversion of cholesterol to testosterone in fetal Leydig’s cells [72]. Considering the negative relationship between levels of testosterone and anxiety-like behavior on the one hand, and the reduction of testosterone release in males with prenatal stress experience on the other hand, it can be concluded that the increased levels of fearful behavior observed in our prenatal infection model of males may be attributed partly to decrease in testosterone, caused by gestational stress. Although the mechanisms responsible for fetal injury induction in these conditions are likely complex, the above evidence suggest that maternally derived IL-6 and corticosterone may directly induce fetal injury and subsequently cause neurobehavioral disturbance of offspring at adulthood. In conclusion, the current investigation indicated that maternal infection during late gestation increases anxiety and depression with increasing age in male offspring, suggesting that time course of an inflammation response or stressor application in various

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