Our study investigates the effects of maternal influenza viral infection on the level of expression and function of 5-HT2A and mGlu2 receptors as a mouse model of schizophrenia. We found that head twitch response, a mouse behavior induced only by hallucinogenic 5-HT2A agonists, was significantly increased in adult mice born to influenza virus-infected mothers. It has been described that mGlu2, and not mGlu3, is the metabotropic glutamate receptor responsible for the effects of the mGlu2/3 agonist LY379268 on the locomotor response induced by the non-competitive NMDA receptor antagonist MK801 (Woolley et al., 2008
); see also (Fell et al., 2008
). Our data demonstrate that the antipsychotic-like behavioral effects of LY379268 are absent in the offspring of influenza virus-infected mothers. The behavioral responses were associated with biochemical alterations in frontal cortex, showing a higher and a lower level of expression of 5-HT2A and mGlu2, respectively, in mice with prenatal exposure to maternal infection. We also found that maternal viral infection leads to an increased cellular response induced by the hallucinogenic 5-HT2A agonist DOI. Thus our results demonstrate that maternal viral infection dysregulates the cellular and behavioral responses that require the 5-HT2A-mGlu2 complex in the adult offspring.
Frontal cortex presents a wide array of cognitive functions, such as learning-reward processing, decision-making, attention and social cognition. From the viewpoint of the model of psychosis paradigm, there is an association between acute psychosis episodes and hyperactivity in frontal cortical regions (Wiesel et al., 1987
; Szechtman et al., 1988
; Parellada et al., 1994
). Several laboratories have reported alterations in frontal cortex of mouse models of maternal viral infection and/or immune challenge, including changes in gene expression (Fatemi et al., 1999
; Fatemi et al., 2002a
; Fatemi et al., 2005
), abnormal cortical neurogenesis (De Miranda et al., 2010
), pyramidal cell atrophy (Fatemi et al., 2002b
; Baharnoori et al., 2009
), and loss of GABA-ergic interneurons (Meyer et al., 2008a
). The behavioral effects of hallucinogenic drugs result from their activity at 5-HT2A receptors expressed by cortical pyramidal neurons (Gonzalez-Maeso et al., 2007
). It has been shown that activation of mGlu2 receptors specifically blocks the cellular and behavioral responses induced by hallucinogenic 5-HT2A agonists (Marek, 2004
; Gonzalez-Maeso et al., 2008
). We have recently reported that, in postmortem prefrontal cortex from young untreated schizophrenic subjects, the 5-HT2A receptor is up-regulated, and the mGlu2 receptor is down-regulated, a pattern that could predispose to psychosis (Gonzalez-Maeso et al., 2008
). Since the same pattern of dysregulated expression was found in frontal cortex of mice born to influenza virus-infected mothers, these results are of considerable importance for future studies to unravel the etiology of schizophrenia and the neurochemical mechanisms responsible for the alterations observed in cortical regions in schizophrenia patients.
It is important to note that many influenza viruses do not optimally replicate or cause disease in mice (Matsuoka et al., 2009
). However, some influenza viruses, including influenza A/WSN/33 (H1N1) virus (Ward et al., 1993
), have been adapted to mice through serial passage, and some highly pathogenic influenza viruses can cause disease and replicate in the respiratory tract and extrapulmonary sites in mice without adaptation (Li et al., 1993
; Takahashi et al., 1995
; Ward, 1995
). Since human influenza viruses replicate mainly in the respiratory tract, a potential factor that may question the validity of the mouse model of maternal viral infection is the presence of influenza virus in fetal brain. Discrepant results have been reported in the literature regarding detection of A/WSN/33 and A/NWS/33 influenza virus strains in the fetus (Aronsson et al., 2002
; Shi et al., 2005
). Our data found that mouse-adapted influenza virus is not detectable in embryo samples from infected mothers. Interestingly, our findings show that influenza A/WSN/33 (H1N1) specific antibody is detected in approximately 5% of all the adult mice (10–14 weeks) whose mothers were infected with influenza virus. Although immature, the fetal immune system is still capable of responding to certain antigenic stimuli, which might account for the antibodies against influenza detected in some of the mice born to influenza virus-infected mothers. Fetuses and neonates are also deficient in several components of inflammatory, innate, and specific immune responses. In humans, maternal antibody transfer to the offspring is mostly transplacental (Leach et al., 1996
). In mice, however, the offspring receive almost all the maternally derived antibody in the colostrum and milk, and this passive antibody typically declines over the first period of life. Although further investigation is necessary, the presence of anti-influenza virus antibodies that we found in some of the adult mice born to infected mothers may be then antibodies passively acquired during lactation. Together with the absence of detectable virus in embryo samples, these data also suggest that the changes observed in the offspring are a consequence of maternal response to viral infection, and not of direct fetal infection by mouse-adapted influenza virus.
Schizophrenia patients show “positive” symptoms (e.g.
, hallucinations, delusions and other thought disorders) and negative symptoms (e.g.
, social withdrawal, apathy and abnormal emotional responses). In addition to the positive and negative symptoms of schizophrenia, the importance of anxiety, depression and cognitive deficits has gained increasing attention. Significant correlations between anxiety symptoms and positive symptoms in schizophrenia have been replicated in several studies (Muller et al., 2004
). Behavior in the open field is used as an anxiety test (Nestler et al., 2002
). In the open field, mice face a conflict between avoidance and exploration of the center which is more aversive than the peripheral area proximal to the walls. It has been shown that measures of behavior in the open field fall into two main categories: 1) time and distance traveled in the center, which may be related to anxiety; and 2) total distance traveled and rearing, which reflect general behavioral activation or exploration. We found that mice born to influenza virus-infected mothers displayed strong deficits in locomotor and exploratory activity, whereas anxiety-like behavior was unchanged.
This reduction of locomotor activity suggests abnormal habituation to a novel environment in mice born to influenza virus-infected mothers. A subset of schizophrenic patients exhibits psychomotor agitation, which includes hyperactivity or increased stereotypic movements (Powell and Miyakawa, 2006
). Locomotor response to novelty has been shown to be higher (Rojas et al., 2007
) and lower (Mukai et al., 2004
) in different knockout mouse models of schizophrenia. However, previous studies have repeatedly demonstrated lower exploratory behavior score in mouse schizophrenia models of perinatal insults (Shi et al., 2003
; Smith et al., 2007
; Meyer et al., 2008b
; Lodge et al., 2009
). Speculatively, the decreased activity that we observed in the adult offspring of influenza virus-infected mothers could be related to negative symptoms of schizophrenia. Earlier reports have shown that mice born to influenza virus-infected mothers spend significantly less time in the center of the squares (Shi et al., 2003
). Here we found that the amount of time spent in the center of the open field was not affected by maternal influenza viral infection. These divergent results may be explicable by strain differences in exploratory behavior between CD1 and BALB/c mice.
Antenatal and postnatal environmental factors have profound implications on the behavior of the adult offspring (Meyer et al., 2009b
). Maternal behavior also alters the offspring epigenome (Weaver et al., 2004
). Thus, litter effects are substantial, and their outcome might not always be independent of the effects of other factors including maternal viral infection. In order to avoid litter effects, our experiments included animals originating from at least three different litters (see Materials and Methods). Although the possibility of litter effects cannot be firmly excluded (Meyer et al., 2009b
), the use of offspring from different mothers, together with the association between biochemical and behavioral results obtained in mice from different litters, suggests that the results described here are consistent with the hypothesis that maternal viral infection affects the level of expression and function of 5-HT2A and mGlu2 receptors.
It has been suggested that prenatal exposure to maternal stress increases the risk of schizophrenia in the offspring (Khashan et al., 2008
; Malaspina et al., 2008
). In rodents, prenatal stress has been shown to induce learning deficits (Lemaire et al., 2000
), as well as alterations in the immune system (Vanbesien-Mailliot et al., 2007
) and feeding behavior (Pankevich et al., 2009
). During shipping, animals are likely to be exposed to noise and vibrations, as well as temperature, humidity, and light-dark changes. Shipping might be then a stressful experience because many of these factors are actually used as independent variables in stress research. In our experiments, timed pregnant mice were shipped on the fifth day of gestation. Although confounding effects from other sources cannot be excluded, mice to be mock- and influenza virus-infected were shipped under the same conditions to minimize a possible bias due to maternal stress.
Some researchers have suggested that maternal infection during pregnancy produces behavioral changes in the offspring that include increased MK801-induced locomotion (Meyer and Feldon, 2009
). We found that the locomotor activity elicited by MK801 in mice born to influenza virus-infected mothers and control mice was indistinguishable. These differences may be due to the dose of MK801 used. The MK801-stimulated locomotor activity was significantly attenuated by the glutamate antipsychotic LY379268 in control mice, but it was not affected in the offspring of influenza virus-infected mothers. In concordance with these effects of maternal viral infection in our mouse behavior models, the density of mGlu2 was reduced in frontal cortex of mice born to influenza virus-infected mothers.
It has been shown that a low dose of the hallucinogen DOI increases locomotor activity, an effect that is absent in 5-HT2A knockout mice (Halberstadt et al., 2009
). In contrast, a high dose of DOI decreases the locomotor activity, an effect that is blocked by a 5-HT2C selective antagonist (Halberstadt et al., 2009
). These findings suggest that 5-HT2A and 5-HT2C receptors exert opposite effects on locomotor activity in mice, and they propose the involvement of 5-HT2A receptor in the locomotor-stimulating effects of DOI. We examined the locomotor activity induced by DOI in mice born to influenza virus-infected mothers and controls. We found that the locomotor activity was decreased by DOI in mice born to mock-infected mothers, whereas it was increased by DOI in mice with prenatal exposure to maternal influenza viral infection. Our data also demonstrate an increased head-twitch response to DOI in mice born to influenza virus-infected mothers (see above). Regulation of c-fos
robustly predicts the head-twitch behavioral response induced by hallucinogenic drugs (Gonzalez-Maeso et al., 2007
). In the present study, we found that the dose-dependent cellular response induced by the hallucinogenic 5-HT2A receptor agonist DOI was significantly different in the adult offspring, with a higher cellular response to DOI in mice born to influenza virus-infected mothers. Concurrently, these cellular and behavioral responses correlate with the higher density of 5-HT2A receptors in mice prenatally exposed to maternal viral infection.
Several laboratories suggest that schizophrenia is a result of neurodevelopmental defects triggered by cytokine-related inflammatory events (Kronfol and Remick, 2000
; Brown et al., 2004b
; Meyer et al., 2009a
; Patterson, 2009
). Thus cytokines and chemokines such as IL-6, CXCL8 (IL-8), IL-10 and TNF-α (Brown et al., 2004b
; Meyer et al., 2006
; Smith et al., 2007
; Meyer et al., 2008b
) have been proposed as responsible for pathways whereby maternal immune activation induces schizophrenia-like behaviors in the adult offspring. Influenza virus infections trigger a profound cytokine and chemokine response at both local and systemic levels, more pronounced with virus strains that appear to be more pathogenic (Kobasa et al., 2007
). Further investigation will be necessary to unravel the role, if any, of cytokines and chemokines on the abnormal behavioral function of the 5-HT2A-mGlu2 receptor complex in schizophrenia murine models of maternal viral infection.
In humans, the onset of schizophrenia typically occurs during late adolescence of early adulthood. Notably, our results demonstrate that alterations in the head-twitch behavioral response are only observed after puberty in prenatally infected mice—a finding that parallels the adult onset of the disease in humans and supports our mouse schizophrenia model of perinatal insult. The antipsychotics currently available are designed only to treat the symptoms of schizophrenia, and not the underlying causes of the disease. Because of this, schizophrenia remains an incurable illness, and treatment of the symptoms with the available antipsychotics typically continues for life. The prevention of psychotic disorders is therefore a worthwhile goal. Remarkably, a prodromal state commonly precedes the acute phase of a first psychotic episode. Recent findings suggest that it is possible to identify young patients at imminent risk of psychosis. Unfortunately, results of several clinical trials that have been published recently demonstrate that the available anti-schizophrenia drugs do not delay or prevent conversion to psychosis in subjects with prodromal symptoms of schizophrenia (McGlashan et al., 2006
; Cannon et al., 2008
; Phillips et al., 2009
). On the other hand, experiments in rodents suggest that prepubertal treatment with atypical antipsychotics such as clozapine and risperidone prevents postpubertal schizophrenia-like alterations induced by prenatal exposure to maternal immune challenge (Meyer et al. 2010
; Piontkewitz et al. 2010
; Piontkewitz et al., 2009
). Our findings may provide a better understanding of the biochemical alterations responsible for the behavioral abnormalities observed in mouse models of prenatal insults, and may lead to the identification of new therapeutic approaches for not only treatment, but also prevention of schizophrenia and other neurodevelopmental psychiatric conditions.