Effects of two commonly found strains of influenza A virus on developing dopaminergic neurons, in relation to the pathophysiology of schizophrenia.

Influenza virus (InfV) infection during pregnancy is a known risk factor for neurodevelopment abnormalities in the offspring, including the risk of schizophrenia, and has been shown to result in an abnormal behavioral phenotype in mice. However, previous reports have concentrated on neuroadapted influenza strains, whereas increased schizophrenia risk is associated with common respiratory InfV. In addition, no specific mechanism has been proposed for the actions of maternal infection on the developing brain that could account for schizophrenia risk. We identified two common isolates from the community with antigenic configurations H3N2 and H1N1 and compared their effects on developing brain with a mouse modified-strain A/WSN/33 specifically on the developing of dopaminergic neurons. We found that H1N1 InfV have high affinity for dopaminergic neurons in vitro, leading to nuclear factor kappa B activation and apoptosis. Furthermore, prenatal infection of mothers with the same strains results in loss of dopaminergic neurons in the offspring, and in an abnormal behavioral phenotype. We propose that the well-known contribution of InfV to risk of schizophrenia during development may involve a similar specific mechanism and discuss evidence from the literature in relation to this hypothesis

Influenza virus promotes neuronal death of dopaminergic neurons in vitro.

<p>Panels (a–c) display characteristic morphology changes of TH stained neurons at baseline (a), 48 h (b) and 72 h (c) post infection; dopaminergic neurons are clearly damaged by the virus, with loss of dendrites and vacuolation of the cytosol. Time curves of TH stained neuronal counts for each viral strain regarding of their morphological state, and therefore probably representing an overestimation of surviving neurons. The A/WSN/33 strain was most toxic at 24 and 48 h. Points represent means of 6–8 independent experiments (two wells per experiment). Standard errors are two small to be displayed on this scale (range 11 to 86). Comparisons were performed by two way ANOVA (treatment and time). ** p<0.001 compared to vehicle.</p

Indirect immunofluorescence for Influenza virus in primary mesencephalic cultures.

<p>Culture dishes infected with New Caledonia-like (H1N1) (panels a–c) or Syndey-like (H2N3) strains (panels d–f), at 24 (a, d), 48 (b, e) and 72 hours (c, f) post inoculation. The two community isolates differed from each in that the New Caledonia-like strain (A/NC-L/99) showed greater affinity at earlier times and more toxic than Sydney-like (A/Sy-L/97) virus.</p

Midbrain gliosis in the adult (p90) offspring of mothers inoculated during pregnancy with control solution (a), or infected with influenza strains A/NC-L/99 (b) or A/WSN/33 (c) during pregnancy day 11.

<p>Sections consecutive to the ones used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051068#pone-0051068-g006" target="_blank">Figure 6</a> were stained for activated glia with antibodies against glial fibrilary acidic protein (GFAP) and photographed at low magnification (100X). There is a clear increase in reactive astrocytes in the two experimental conditions, when compared to controls.</p

Influenza infection results in NFκB activation in vitro.

<p>Panels (a) and (b) show NFκB staining in primary cultures following inoculation of A/WSN/33 (a) or PBS solution (b). Cultures (11 day in vitro) were inoculated with PBS, A/WSN/33, A/NC-L/99 (H1N1), A/Sy-L/97 (H2N3) influenza strains as described in the text, fixed, immunostained for NFκB and neurons were counted at times specified in panel (c). Cell bars represent means of 6–8 independent experiments (two wells per experiment). Error bars are SEM. Comparisons were performed by two way ANOVA (treatment and time). ** p<0.001 compared to control (PBS solution).</p

Morphological changes in embryos from mothers infected with influenza Virus strains sacrificed 3 days following inoculation of control solution (a,d), or infection with influenza A viral strains A/NC-L/99 (b,e) or A/WSN/33 (c,f) on pregnancy day 11.

<p>Top panels display low magnification (X200) of the area of the substantia nigra stained with Nissl. Bottom panels show electron micrographs (X24,000) obtained from the same specimens. Infection with influenza results in significant swelling of midbrain embryonic neurons. Arrowheads indicate vacuolar dilatations.</p

Behavioral assessments in offspring of infected mothers.

<p>Means are provided for for exploratory behavior, rearing and and anxiety levels in the Open Field, exploratory behavior of a novel object, and performance on an elevated cross maze test of young (post natal day 30) and adult (post natal day 90) offspring of mothers inoculated with control solution (PBS) or infected with influenza A strains A/WSN/33 or A/NC-L/99. A separate cohort of adult offspring was tested for an object recognition task; time spent exploring the familiar and non-familiar object was computed and the difference (Δ) is reported. Values represent means ± SEM. Bold values in grayed cells represent p<0.01 compared to control. Italic values in grayed cells represent p<0.05 compared to control. Statistics represent ANOVA followed by Kruskal Wallis post hoc comparisons for means.</p

Midbrain dopaminergic nuclei in the adult (p90) offspring of mothers inoculated during pregnancy with control solution (a,b,c), or infected with influenza strains A/NC-L/99 (d,e,f) or A/WSN/33 (g,h,i) during pregnancy day 11.

<p>From left to right, panels show increasing magnification of tyrosine hydroxilase immunostaining (100X, 400X, 1000X). There is a clear loss of stained neurons in the exposed animals, and at higher magnification a dystrophic appearance of surviving neurons is evident. Panel (j) displays mean ± SEM of stereological counts of dopaminergic (TH positive) neurons throughout the entire brainstem (n = 4/group). * p<0.05 compared to control. The insert displays the results discriminating between substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) in a separate set of 4 animals for each viral strain.</p

Summary of animals used in the in vivo experiments.

<p>Summary of animals used in the in vivo experiments.</p

Influenza infection causes neuronal apoptosis in vitro.

<p>Panels a and b show TUNEL staining in primary cultures following inoculation of cultures (11 day in vitro) with either PBS solution (a) or A/WSN/33 (d) at 200X. Cultures were fixed, stained for TUNEL and counted at times specified on the x axis of panel (c). Cell counts bars represent means of 6–8 independent experiments (two wells per experiment). Error bars are SEM. Comparisons were performed by two way ANOVA (treatment and time). ** p<0.001 compared to control (PBS solution).</p