Prenatal lipopolysaccharide exposure increases anxiety-like behaviors and enhances stress-induced corticosterone responses in adult rats

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Abstract

Maternal infection during pregnancy may affect fetal brain development and lead to neurological and mental disorders. Previously, we used lipopolysaccharide [LPS, 33 μg/kg, intraperitoneal injection] exposure on gestation day 10.5 to mimic maternal bacterial infection in rats and found reduced dopaminergic and serotoninergic neurons in the offspring. In the present study, we examined the anxiety and stress responses of the affected offspring and the neurophysiological changes in their brains. Our results show that LPS rats displayed more anxiety-like behaviors and heightened stress responses. Dopamine (DA) in the nucleus accumbens and serotonin (5-HT) in the medial prefrontal cortex and the hippocampus were significantly reduced in LPS rats. Their glucocorticoid receptors in the dorsal hippocampus and the 5-HT1A receptors in the dorsal and ventral hippocampus were also reduced. In addition, chronic but not acute fluoxetine treatment reversed the behavioral changes and increased hippocampal 5-HT1A receptor expression. This study demonstrates that LPS exposure during a critical time of embryonic development could produce long-term reduction of DA and 5-HT and other neurophysiological changes; such alterations may be associated with the increases in stress response and anxiety-like behaviors in the offspring.

Highlight

► Maternal immune activation, if occurs during the critical window for DA and 5-HT neurogenesis, could increase the anxiety-like behaviors and stress responses in offspring.

Introduction

Maternal infection during pregnancy can increase the risk of abortion, preterm delivery, neurodevelopmental abnormality, mental retardation, and psychiatric disorders in offspring (Dammann and Leviton, 1998). A number of theories of the etiology of mental disorders suggest a disturbance of the fetal nervous system during pregnancy as a major factor (Meyer et al., 2005, Bransfield et al., 2007). During fetal development every area of the brain develops at a specific time. An area that does not develop fully in the assigned period is often left with a functional deficit. Thus, the timing of the perturbation in neurodevelopment may be critical in determining whether and what type of mental illness will occur. Empirically an association between maternal infection during mid-pregnancy and an increased risk of developing schizophrenia and autism in offspring has been found in many epidemiological studies (Atladóttir et al., 2010, Mednick et al., 1988, O’Callaghan et al., 1994, Brown et al., 2004). It is less clear whether maternal infection can also increase the risk of development of mood disorders in the offspring. Both the Helsinki cohort, which originally identified the association between maternal influenza infection and an increased risk of schizophrenia, and a Dublin cohort exposed in the uterus to A2 influenza epidemic showed a significantly increased risk of the offspring developing affective disorders (Cannon et al., 1996, Machón et al., 1997). These findings support the hypothesis of a possible neurodevelopmental contribution to the origin of some forms of mood disorder. However, a recent long-term cohort study in the United Kingdom failed to find any association between prenatal viral infection and depression later in life (Pang et al., 2009). On the other hand, preclinical studies seem to provide more consistent results on the link between maternal infection and behavior changes relevant to major affective disorders in offspring. It has been shown that mice with prenatal exposure to LPS or viral mimic polyriboinosinic–polyribocytidylic acid (polyI:C) exhibit signs of heightened anxiety (Hava et al., 2006, Meyer et al., 2006).

Maternal bacterial infection is though to affect the developing brain via the induction of pro-inflammatory cytokines, which could cross the placenta barrier and regulate the developing neurons (Golan et al., 2005, Hava et al., 2006). In preclinical studies, bacterial endotoxin LPS is often used to induce the defensive pro-inflammatory cytokines. LPS injection in gravid dams could significantly elevate tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in the amniotic fluid of the fetus (Gayle et al., 2004). In addition, cytokines TNF-α and IL-1β have been shown to regulate survival of embryonic dopamine (DA) and serotonin (5-HT) neurons (Jarskog et al., 1997). Our previous studies also demonstrated that a single LPS injection (33 μg/kg) on gestation day 10.5 (E10.5) when DA and 5-HT neurons first emerge (Specht et al., 1981, Lauder, 1990) can cause significant loss of DA neurons in the substantia nigra and 5-HT neurons in the dorsal raphe (Wang et al., 2009) in the offspring. In light of the association of DA and 5-HT in the mesolimbic system with the control of emotional and stress-related behaviors (Ressler and Nemeroff, 2000, Pezze and Feldon, 2004, Lowry et al., 2008), it is highly plausible that DA and 5-HT deficiencies might produce behavioral abnormalities related to mood regulation in offspring.

In the present study, we examined the effects of prenatal LPS exposure during the critical time of DA and 5-HT neurogenesis on anxiety-like behaviors and stress responses in the adult offspring. In addition to behavior tests, we also examined DA and 5-HT in related brain areas and the 5-HT1A receptor in the hippocampus. In light of the enhanced stress response found in prenatal LPS-exposed rats, the hypothalamic–pituitary-adrenal (HPA) axis feedback mechanisms and glucocorticoid receptors in related brain areas were also examined. Finally, we administrated both acute and chronic antidepressant fluoxetine (FLX) to evaluate its effects on reversing behavioral abnormalities in LPS rats. The results show that prenatal LPS exposure increased anxiety-like behaviors and enhanced stress responses in offspring and that chronic FLX treatment can effectively reduce the anxiety-like behaviors. These findings support the hypothesis that perturbation in neurodevelopment could contribute to the development of mood disorders in offspring. Prenatal LPS exposure could be used as an animal model for studying anxiety disorders.

Section snippets

Animals

Gravid Sprague–Dawley rats (6 weeks old, 230–260 g, BioLASCO Taiwan Co., Ltd., Taipei) and their offspring were used in this study (dam, n = 43, offspring, n = 350). The gravid rats were housed individually and kept at an ambient temperature of 24 °C and a 12-h light/dark cycle (light on 6:00–18:00). Food and water were provided ad libitum. After 2 days of habituation to the animal housing facility, pregnant rats were given a single ip injection of LPS (20,000 U/kg, equivalent to 66 μg/kg, L8274,

Maternal isolation stress

On P21, the pups were isolated from their dams for at least 30 min in a different room (n = 8/group). Then the pups were anesthetized by isoflurane, and blood samples were collected from the heart before perfusion. Plasma corticosterone was measured by enzyme-linked immunoabsorbent assay kits (Assay Designs).

Prenatal LPS-exposed rats show increased anxiety

To assess anxiety-like behaviors in prenatal LPS-exposed rats, we conducted three different behavioral tests, open field, elevated plus maze, and novelty-induced hypophagia tests. To rule out any possible movement deficits that could affect the results and hence interpretation of the behavior tests, we first used a small open field box to examine the general activity of the rats. In the small open field, the LPS rats showed no difference in total travel distance and movement velocity compared

Discussion

In the current study, we have demonstrated that prenatal LPS exposure increased anxiety-like behaviors in offspring in all three behavior tests we employed (Fig. 2). A recent study by Kirsten et al. (2010) shows that adult male offspring from Wistar rats injected with LPS (100 μg/kg, serotype 0127:B8) on gestation day 9.5 (E9.5) have reduced social behavior. The injection time was chosen to target cerebral organogenesis, particularly neural plate formation. That time point is slightly earlier

Financial disclosures

The authors, Yu-Lung Lin, Shu-Yi Lin, and Sabrina Wang report no biomedical financial interests or potential conflicts of interest.

Acknowledgments

This study was supported by a research grant to S.W. from the National Science Council of Taiwan (Grant number NSC97-2314-B-400-002-MY3). We thank Dr. Chung-Shi Yang and Mr. Te-Haw Wu for their assistance with the HPLC experiments.

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