Defective corticogenesis and reduction in Reelin ... - Nature

Molecular Psychiatry (1999) 4, 145–154  1999 Stockton Press All rights reserved 1359–4184/99 $12.00

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Defective corticogenesis and reduction in Reelin immunoreactivity in cortex and hippocampus of prenatally infected neonatal mice SH Fatemi1,2, ES Emamian1, D Kist1, RW Sidwell3, K Nakajima4, P Akhter1, A Shier1, S Sheikh1 and K Bailey3 Departments of 1Psychiatry; 2Cell Biology and Neuroanatomy, Division of Neuroscience Research, University of Minnesota Medical School, Box 392, 420 Delaware St SE, Minneapolis, MN 55455, USA; 3Institute for Antiviral Research, Utah State University, Logan, Utah, USA; 4Department of Molecular Neurobiology, Institute of DNA Medicine, Jikei University, School of Medicine, Minato-Ku, Tokyo, 105–8461, Japan Recent reports indicate an association between second trimester human influenza viral infection and later development of schizophrenia. Postmortem human brain studies also provide evidence for reduction in Reelin mRNA (an important secretory protein responsible for normal lamination of the brain) in schizophrenic brains. We hypothesized that human influenza infection in day 9 pregnant mice would alter the expression of reelin in day 0 neonatal brains. Prenatally-infected murine brains from postnatal day 0 showed significant reductions in reelinpositive cell counts in layer I of neocortex and other cortical and hippocampal layers when compared to controls. Whereas layer I Cajal–Retzius cells produced significantly less Reelin in infected animals, the same cells showed normal production of calretinin and nNOS when compared to control brains. Moreover, prenatal viral infection caused decreases in neocortical and hippocampal thickness. These results implicate a potential role of prenatal viral infection in causation of neuronal migration abnormalities via reduction in Reelin production in neonatal brains. Keywords: prenatal; influenza; infection; Reelin; schizophrenia; mice

Introduction Schizophrenia is a severe brain disorder which affects 1% of the world population today.1 Early investigators had suspected a potential biological origin for this disorder.2,3 Recent reports have indicated neuropathologic and neurochemical abnormalities in postmortem brains of patients with schizophrenia.4–9 More specifically, abnormal translocation in NADPH-diaphorase positive cells in frontal and temporal cortices hint at neurodevelopmental causes for schizophrenia.4,5 The neurodevelopmental etiology of schizophrenia has also been supported by recent epidemiologic reports10 showing an association between maternal second trimester human influenza viral infection and later increased risk for development of schizophrenia.10 Despite preponderance of positive epidemiologic data, experimental evidence supporting such a claim is lacking. A recent report11 showed a mild increase in pyramidal cell disarray of dorsal hippocampus in murine neonates born to mothers exposed to human influenza

Correspondence: SH Fatemi, MD, PhD, University of Minnesota Medical School, Division of Neuroscience Research, Box 392 UMHC, 420 Delaware St SE, Minneapolis, MN 55455, USA. E-mail: fatem002얀gold.tc.umn.edu Received 30 November 1998; accepted 17 December 1998

virus on day 13 of pregnancy. Further evidence from our laboratory showed that prenatal viral infection on day 9 of pregnancy causes alterations in nNOS and SNAP-25 levels in day 0 neonatal brains.12,13 We hypothesized that prenatal human influenza viral infection12 on day 9 of pregnancy in C57BL/6 mice may alter production of Reelin by Cajal–Retzius cells causing subsequent morphologic or synaptic changes in brains of day 0 neonatal mice. Reelin is a secretory glycoprotein with relative molecular mass of about 400 kDa14 and encoded by a long mRNA of about 12 kilobases.15,16 The aminoterminus of Reelin is 25% identical to that of F-spondin,16 a protein secreted by the floor plate of the spinal cord and thought to regulate the adhesion and extension of commissural axons17 and to tenascin.18 Reelin RNA is first detectable in the mouse embryonic brain on day 9.5.19 It then increases in concentration up to early postnatal days and then declines to adult levels. The first cells producing Reelin are the pioneer Cajal–Retzius neurons which begin differentiation as early as day 9.5 in embryonic mouse brain;20 these are transient neurons that act as pathfinders and help in the early laminar organization of the cortex.16,19,20 The reeler mutant mouse exhibits widespread morphological abnormalities in various cortical structures including abnormal positioning of neurons and aberrant orientation of cell

Prenatal viral infection and Reelin SH Fatemi et al

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bodies and fibers.21,22 In the cerebral cortex of the reeler mouse, neurons destined to form the subplate zone occupy ectopic positions in superficial cortical layers. Additionally, neurons developed later which are destined to form the cortical plate, fail to bypass previously generated neurons.22 Thus, an inverted pattern of cortical development takes place in the mutant mice. There is a striking similarity in migration abnormalities involving the NADPH-diaphorase positive cells in schizophrenic brains and the reeler phenotype.4,5 The human reelin cDNA has been cloned and maps to region 22 on the long arm of chromosome 7.23 Two recent reports showed a 40–50% decrease in reelin mRNA in brains of schizophrenic patients.24,25 We investigated the role of human influenza viral (HI) infection prenatally in C57BL/6 mice.12,13 Intranasal infection of day 9 pregnant mice with 10−5 dilution of HI (H1N1) caused sublethal infection in pregnant mice.12,13 Day 0 neonatal brain sections from infected and sham-infected groups were prepared for immunocytochemistry.12,13 Immunocytochemical localization12,13 of Cajal–Retzius (CR) cells containing Reelin was accomplished using a specific monoclonal antibody (CR-50) against mouse reelin.20 Here we show that prenatal viral infection through decrease in production of Reelin, may cause abnormal neocortical cerebral development.

Methods and materials Animals and viral infection Female 12–14-week-old specific pathogen-free C57BL/6 mice were obtained from Simonsen Laboratories (Gilroy, CA, USA). For initial virus titration studies, the animals were quarantined 24 h prior to use and maintained on Wayne Lab Blox and tap water. After being infected, their drinking water contained 0.006% oxytetracycline (Pfizer, New York, NY, USA) to control for possible bacterial infection. In experiments using pregnant C57BL/6 mice, the female mice were bred with male mice of the approximate same age after at least 1 week of quarantine. Initial pregnancy was determined by observation of vaginal plug in the animals following breeding. Influenza A/NWS/33 (H1N1) was obtained from RW Cochran (University of Michigan, Ann Arbor). A virus pool was prepared in Maden Darby canine kidney (MDCK) cells; the virus was ampuled and frozen at −80°C until used. Mice were anesthetized by intraperitoneal (i.p.) injection of approximately 167 mg kg−1 ketamine (Phoenix Scientific, St Joseph, MO, USA), and while under the effects of anesthetic were inoculated by intranasal instillation (i.n.) with 90 ␮l of virus. The virus was diluted 10−4, 10−4.5, 10−5, or 10−6 times; these dilutions were based on previous titrations performed in other mouse species. A total of 10 mice were exposed to each virus dilution; occurrence of death was noted daily for 21 days in five of these animals; the remaining mice were killed on infection day 7 and their lungs removed and assigned a consolidation score of 0 (normal) to 4 (entire lung displaying typical plum coloration associated

with influenza virus infection). Each lung was also weighed to determine if weight gain due to consolidation occurred and then each was homogenized to a 10% (wt/vol) suspension in minimum essential medium (MEM) containing Earle’s balanced salt solution, 0.1% NaHCO3, 1% sorbitol, and 50 ␮g gentamicin ml−1. Each lung homogenate was then diluted through a series of tenfold dilutions and assayed for infectious virus in triplicate in 96-well microplates containing a 24-h monolayer of MDCK cells. Virusinduced cytopathic effect determined microscopically was used as the infectivity end point. Data were expressed as log10 cell culture infectious doses (CCID50) ml−1 by the method of Reed and Muench.26 By this titration, it was determined that at a dilution of 10−5 none of the mice died of the infection, but displayed a mean lung consolidation score of 1.4, a mean lung weight of 258 mg (51% higher than normal lungs), and had a mean virus titer of 105,25 CCID50 ml−1, indicating that a moderate but sublethal infection had been induced. This was the virus dose selected for use in the pregnant mouse study. Pregnant mice 9 days after breeding were exposed i.n. to a 10−5 dilution of virus by the method described below. As controls, additional pregnant mice were exposed to sterile virus diluent in the same manner as the infected animals. Three mice from each group, killed on infection day 7, had their lungs removed and processed as in the virus titration. In the infected group, the mean lung score was 2.0, the mean lung weight was 263 mg, and the mean virus titer was 104,4 CCID50 ml−1. In the control group, no lung consolidation score was seen, the mean lung weight was 170 mg, and no virus was detected in the lungs. All the animals withstood the anesthesia and i.n. procedure in a satisfactory manner. Immunohistochemistry Pregnant mice were allowed to deliver pups. The day of delivery was considered day 0. Groups of infected (n = 12 for reelin experiments, n = 8 for calretinin experiments, n = 4 for nNOS experiments) and shaminfected neonates (n = 9 for reelin experiments, n = 5 for calretinin experiments, n = 5 for nNOS experiments) were deeply anesthetized. Brains were removed from skull cavities and immersed in phosphate-buffered 4% paraformaldehyde (pH 7.4) for 7 days at 4°C. Coronal sections (10 ␮m) were cut on an IEC-minitome cryostat (IEC, Needham Heights, MA, USA) and placed on subbed slides. Alternatively, cryopreserved brains were snap-frozen by immersion in liquid nitrogen and stored at −85°C for future use. All infected and sham-infected sections of the cortex and hippocampus were level-matched using the atlas of embryonic mouse brain.27 Sections were taken from a minimum of three levels of cortex and hippocampus in experimental and control mice. Sections were warmed to room temperature (RT), dried, postfixed in acetone for 10 min and redried at RT; they were subsequently washed three times at RT in phosphate-buffered saline containing 0.2% Triton X-100 (PBS, 10 mM


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sodium phosphate, 150 mM NaCl, pH 7.4). Later, sections were immersed for 30 min at RT in PBS containing 0.2% Triton X-100 and 3% normal goat serum. The sections were incubated with the following antibodies: mouse CR-50 (anti-Reelin) monoclonal antibody, 1:10;19 rabbit polyclonal anti-nNOS antibody 1:1000 (Santa Cruz, CA, USA); rabbit anti-calretinin polyclonal 1:5000 (SWANT, Bellizona, Switzerland); rabbit anticalretinin polyclonal 1:1000 (Chemicon, Temecula, CA, USA). Following a 24–72 h preincubation with primary antibodies at 4°C and multiple washes in PBSTX-100 (0.2%), 5 nm gold-conjugated to appropriate secondary antibodies (Goldmark, NJ, USA) were added to the slides at a dilution of 1:100, (except for 1:400 with Chemicon anticalretinin) for 1 h at RT (gold-conjugated goat anti mouse IgG against reelin (CR50); goldconjugated goat anti rabbit IgG against nNOS; gold-conjugated goat anti rabbit IgG against calretinin). Subsequently, silver (Goldmark, NJ, USA) enhancement was carried out on all slides, including control sections (without primary antibody) at a dilution of 1:1 of developer and enhancer for 17–25 min at RT. Reaction times for silver deposition were always identical in control and infected brain sections. The reactions were terminated by washing the slides with distilled water. Slides were then dehydrated through a graded alcohol series and xylene and coverslipped in permount before examination with a Nikon Labophot-2 microscope (Fryer, Bloomington, MN, USA) under bright light field. Cell counting and area measurements Reelin-positive cells were counted blindly by two individuals and cells were counted based on immunostaining and determination of the morphology of these cells (Figure 1). Cell counts were performed using the Micro-bright field Stereo Investigator software (Burlington, VT, USA). Generally, large cells with horizontal dendrites present in layer I consisted of Cajal– Retzius cells. Additionally, similar cell types were observed and counted in the marginal zone of the hippocampus. Other Reelin-positive cells19 which appeared smaller than Cajal–Retzius cells were localized throughout cortical and hippocampal layers and counted. Similar large cells resembling CR cells which expressed nNOS and calretinin were also identified and counted in the cortical layer I of control and infected brains. Moreover cresyl-violet stained CR cells, pyramidal and nonpyramidal neurons were identified and counted. The areas were measured using the Micro-bright field Stereo Investigator software and included: cortical layer I, cortical layers II–VI and intermediate zone (presumptive white matter), and hippocampal layers (marginal zone, stratum radiatum, stratum pyramidale, and stratum oriens) and subjacent intermediate zone, and total unilateral brain hemisphere. Measurements were made using cresyl violet-stained sections selected from three rostrocaudal areas of the brain approximating to the level of septodorsal hippocampus (level 1), mid septotemporal hippocampus (level 2) and temporoventral hippocampus (level 3). These measurements

Figure 1 Reelin-positive Cajal–Retzius cells are seen in cortical layer I and hippocampal marginal zone in prenatallyinfected (b, d, f, h) and sham-infected brains (a, c, e, g). At ×20 and ×60 magnifications, Cajal–Retzius cells lie horizontally in layer I of control brain (a and c respectively). At the same magnifications, two horizontally located Cajal–Retzius cells are seen (b and d). At ×6.4 and ×40 magnifications, higher numbers of Cajal–Retzius cells can be demonstrated in the hippocampus of sham-infected mice (e, g) as compared to infected (f, h) respectively.

were obtained from a minimum of three sections per brain from control (n = 9) and infected (n = 12) mice. Mean areas in mm2 ± SD were obtained and subjected to statistical analysis. Finally, cell density values for reelin-positive CR and non-CR cells were obtained by dividing the cell count by mean of each area measured from cresyl violetstained neighboring sections and expressed as cell counts per mm2.


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Statistical analysis Statistical analysis of data was performed using InStat GraphPad Software (NY, USA) and consisted of the following: (1) Analysis of data in Figure 2a, c, d, e and f by Bartlett’s test showed that variances were not equal, thus data were then subjected to Kruskal–Wallis nonparametric ANOVA followed by Dunn’s post test to obtain statistically significant differences between groups; (2) Analysis of Figure 2b data, by Bartlett’s test, showed that variances were equal. Thus one-way ANOVA followed by Tukey–Kramer post test was performed to establish significance; (3) In analysis of data for Figure 2 g, h, Figure 3 and Table 1, F testing showed that variances were not equal. Thus data were subjected to Mann–Whitney non-parametric test to establish significance; (4) Finally, analysis of data for Table 2 used an unpaired t-test to establish significance.

Results Quantitation of Reelin-positive cells in cerebral cortex and hippocampus Reelin-positive cells were identified and counted in layer I of the cerebral cortex and the marginal layer of hippocampus and in the cortical and hippocampal layers and subjacent intermediate zone. Reelin-positive cells were observed in both prenatally-infected and sham-infected neonatal brains (Figure 1). In layer I of sham-infected cerebral cortex, Reelin-positive cells showed morphologic characteristics of CR cells; these cells appeared larger than pyramidal cells with intracellular staining surrounding an oval or round unstained nucleus. CR cells exhibited horizontal extensions and were identifiable predominantly in layer I of cerebral cortex and hippocampal marginal zone in infected and sham-infected brains. Moreover, smaller Reelin-positive cells, presumably GABA-containing neurons and interneurons19,28 could be seen scattered throughout cerebral cortical layers II–VI, as well as in the developing hippocampal layers in both infected and sham-infected brains (Figure 1). Quantitative assessment of Reelin-positive cells showed significant differences in the number of these cells in several areas of the prenatally-infected neonatal brains as compared to controls (Figure 2 a–f, P ⬍ 0.0001, ANOVA). Prenatally-infected cortical layer I and hippocampal marginal zone showed significant reductions in Reelin-positive CR cell counts in mid septotemporal (MST) and temporal ventral (TV) brain areas (rostrocaudal positions 2 and 3 respectively) when compared to sham-infected brains (for cortical layer I, MST, P ⬍ 0.01, TV, P ⬍ 0.001; for hippocampal marginal zone, MST, P ⬍ 0.01 and TV brain P ⬍ 0.05). The septodorsal (SD) brain cell counts (rostrocaudal position 1) did not differ significantly between the two groups (Figure 2a and 2b). Reelin-positive cell counts in cortical layers II–VI, again showed statistically significant reductions in infected MST and TV brain levels (cortical layers II–VI, MST P ⬍ 0.001; TV brains P ⬍ 0.01) and in all hippocampal layers except intermediate zone of TV brains (hippocampus P ⬍ 0.001)

(Figure 2 c, d). The other areas of the brains showed reductions, but these were not statistically significant (Figure 2 c, d). When Reelin-positive cell counts for combined cortical layer I and hippocampal marginal zone and all other cortical and hippocampal layers were analyzed, statistically significant differences were observed in prenatally-infected brains as compared to controls (Figure 2 e, for cortical layer I and hippocampal marginal zone in MST and TV brains, P ⬍ 0.001 and P ⬍ 0.001 respectively; Figure 2f, for other layers in cerebral cortex and the corresponding layers of hippocampus in MST and TV brains, P ⬍ 0.01 and P ⬍ 0.001 respectively). Finally, when all Reelin-positive cell counts were combined according to brain compartment ie as total of cells in neocortical layer I and hippocampal marginal zone (Figure 2g), total of layers II–VI in neocortex and the corresponding layers of hippocampus (Figure 2h), regardless of rostrocaudal position of brain, infected values were statistically significantly reduced (P ⬍ 0.0001 layer I and marginal zone; P ⬍ 0.0001 all other layers) as compared to controls. To control for the potentially confounding factor of a general reduction in tissue mass, an additional set of calculations was performed. In this analysis, Reelinpositive cell counts were converted to cell density values. Calculation of Reelin-positive cell density values per mm2 showed statistically significant reductions in hippocampal (P ⬍ 0.0007) and neocortical (P ⬍ 0.0008) regions in prenatally infected brains when compared to control values (Table 1). Cerebral cortical layer I cell densities were unchanged in experimental brains as compared to controls (Table 1). This is probably due to a larger and significant decrease (P ⬍ 0.0001) in cerebral cortical layer I area (39% decrease) observed in prenatally-infected brains (Table 2), as opposed to others measured; thus artifactually increasing the density values for infected cortical layer I. Moreover, area measurements of cerebral cortical layers II–VI and intermediate zone and total unilateral hemispheres showed statistically significant reductions in infected day 0 brains as compared to controls (Figure 4, Table 2).19 The hippocampal values, despite a large reduction, were not statistically significant (Table 2). Quantitation of Calretinin, nNOS and Reelin-positive CR cells in layer I of cerebral cortex We further investigated whether the reductions in Reelin-positive cell number and density were due to changes in synthesis or degradation of Reelin protein or cell death. It has been known that CR cells do produce a number of other markers both during development and in adult life.29–32 Calretinin immunoreactive CR cell count in layer I of cortex did not differ between experimental and control brains (Figure 3). Additionally, nNOS immunoreactive cell counts did not differ significantly in cerebral cortical layer I of infected and control brains (Figure 3). Moreover, prenatal viral infection in this study occurred on day 9 of pregnancy, approximately 0.5–2 days prior to genesis of CR cells on day 9.519 to 1120,32 of pregnancy suggest-


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Figure 2 The values expressed on the y-axis are total reelin-positive cell counts per single brain hemisphere. The x-axis values show approximate locations of sections sampled from septodorsal, midseptotemporal and temporoventral areas of brain (rostrocaudal positions 1, 2 and 3 respectively). The number of mice used in prenatally infected and sham-infected groups were n = 12 and n = 9 respectively. The cell counts are reduced significantly in positions 2 and 3 of infected (I) cortical layer I (P ⬍ 0.01; P ⬍ 0.001) and hippocampal marginal zone (P ⬍ 0.01; P ⬍ 0.05) (a and b respectively) and other infected cortical (P ⬍ 0.001, P ⬍ 0.01) and hippocampal (P ⬍ 0.001) layers (c and d, except position 2 of d) when compared to controls (C). Combination of cell counts in total cortical layer I and hippocampal marginal zone (e) and total additional cortical and hippocampal layers (f) show significant reductions in infected positions 2 and 3 (P ⬍ 0.001, P ⬍ 0.001 layer I and marginal zone respectively; P ⬍ 0.01, P ⬍ 0.001 other cortical and hippocampal layers respectively). Finally global reelin-positive cell counts in hemispheric cortical layer I and hippocampal marginal zone (P ⬍ 0.0001) and other layers (P ⬍ 0.0001) also showed statistically significant reductions in the infected mice (g and h).


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Figure 3 The top panel shows three graphs depicting the hemispheric immunoreactive Cajal–Retzius cell counts in layer I of cortex in prenatally-infected (I) and sham-infected (C) animals, using three markers of Cajal–Retzius cells ie, reelin (CR 50), calretinin, and nNOS. There is a significant reduction in the number of reelin-positive CR cells in the infected brains (n = 12, mean ± SD, 72.2 ± 25.6) vs control brains (n = 9, mean ± SD, 114.5 ± 46.2, P ⬍ 0.0001). There are, however, no statistically significant differences in the number of calretinin positive (infected n = 8, mean ± SD, 32.8 ± 4.2, control n = 5, mean ± SD, 33.3 ± 8.6); and nNOS positive (infected n = 4, mean ± SD, 12.1 ± 4.9, control n = 5, mean ± SD, 16.4 ± 7.9) CR cells between prenatally infected and sham-infected animals. The lower panel shows light micrographs of layers I–II in coronal sections of prenatally-infected (b, d, f) and sham-infected cortex (a, c, e).

Table 1 Comparison of reelin-positive cell density expressed as cells per mm2 in prenatally-infected and shaminfected day 0 neonatal brains Animals

Cerebral cortical layer I

Sham-infected 211.1 ± 77.1 (n = 9) Infected 212.5 ± 75.5 (n = 12) %⌬ –

Cerebral Hippocampus cortex (layers (all layers) II–VI) 74.0 ± 60.8

166.2 ± 79.8

31.4 ± 21.9*

107.9 ± 51.2**

↓ 57.5%

↓ 35%

Values have been expressed as mean ± SD. * P ⬍ 0.0008 (Mann–Whitney test). ** P ⬍ 0.0007 (Mann–Whitney test).

ing that prenatal viral infection may not have reduced the number of CR cells since cortical layer I calretinin and nNOS-counts between the two groups would have to also be altered. Moreover, cresyl violet-stained CR cell counts in layer I of infected brains did not differ significantly when compared to control brains (Fatemi et al; unpublished data). Collectively, these data indicated that cell death may not account totally for changes in Reelin-positive cell number and density in

infected brains, but point to abnormal production of Reelin in infected neurons potentially due to either decreased synthesis or increased degradation of the Reelin molecule (Figure 3). Abnormal cerebral cortical development in prenatally infected mice Area measurements in cerebral cortex, hippocampus and unilateral brain hemispheres were decreased in the infected neonatal brains (Figure 4, Table 2). Specifically, there were statistically significant decreases in cerebral cortical layer I (P ⬍ 0.0001, ⬵ 39% decrease), in overall cerebral cortical layers II–VI including the intermediate zone (P ⬍ 0.0015, ⬵ 27.2% decrease), and in total unilateral brain hemisphere (P ⬍ 0.0001, ⬵ 26.5% decrease) when compared to control values (Table 2). Additionally, there was a 18.1% decrease in hippocampal area in infected brains which did not attain statistical significance when compared to control value (Table 2). A review of infected temporal-ventral brains showed increase in pyramidal cell density of 47% (P ⬍ 0.031) in cerebral cortex (data not shown). Estimation of pyramidal cell nuclear size in infected temporal-ventral cerebral cortex showed statistically significant decrease of 41% (P ⬍ 0.037) in all cortical layers (data not shown).


Table 2

Comparison of area measurements (mm2) between prenatally-infected and sham-infected brain areas

Animals

Cerebral cortical layer I

Cerebral cortex (layers II–VI) and 1Z a

Hippocampus (all layers) and 1Z a

Total unilateral brain hemisphere

Control (n = 9) Infected (n = 12) %⌬

0.56 ± 0.07

4.44 ± 1.08

0.99 ± 0.33

10.3 ± 1.58

0.34 ± 0.09*

3.23 ± 1.26**

0.81 ± 0.35

7.57 ± 1.97*

↓ 39.3%

↓ 27.2%

↓ 18.1%

↓ 26.5%

Values have been expressed as mean ± SD. * P ⬍ 0.0001 (unpaired t-test). ** P ⬍ 0.0015 (unpaired t-test). IZ a = Intermediate zone.

Figure 4 Cresyl violet-stained coronal sections of cortical layers I–VI and the subjacent intermediate zone in prenatally-infected (b, d and f) and sham-infected brains (a, c, e). At lower magnification (×10) there is clear reduction in width of layer I and other layers of cortex in infected brains (compare b to a). The reductions in area measurements for infected cortical layer I and layers II–VI and WM and unilateral brain hemispheres were statistically significant as compared to control values (P ⬍ 0.0001 cortical layer I; P ⬍ 0.0015 cortical layers II–VI and WM; P ⬍ 0.0001 unilateral hemisphere). Despite an obvious 18.1% reduction in infected hippocampal area, the value obtained was not statistically significant when compared to control. At higher magnifications (×16 c and d; ×40 e and f), the lamination pattern becomes difficult to ascertain and indistinct in prenatally infected brains (d vs c) as compared to controls.

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Discussion Prenatal human influenza viral infection in utero on day 9 of pregnancy by a neurotropic strain of HI virus (H1N1), caused global reductions in the production of Reelin in cerebral cortex and hippocampus of day 0 neonatal brains. Moreover, analysis of brains showed abnormal neuronal cell development and migration in the cerebral cortex of experimental brains. Reelin production in reeler mutant mouse is defective.16 There are, however, quantitative differences between the types of mutations that affect the reelin gene and its synthetic product Reelin.30 Thus, in the original reeler mutation, no Reelin product could be identified in cerebral cortex, hippocampus and cerebellum of reeler mutant brains.20,33 Alternatively, a truncated Reelin protein is produced but not secreted in the ‘Orleans’ reeler mutation.34 Thus variable expression of Reelin could take place secondary to the nature of a mutation or a biochemical defect. A recent report35 indicated that CR cells of the cerebral cortex express receptors for the neurotrophin brain-derived neurotropic factor (BDNF) which causes subsequent decrease in production of Reelin during early postnatal development. Acute BDNF stimulation of cortical neuron cultures and/or overexpression of BDNF in the brains of transgenic mice cause a dose-dependent reduction in Reelin expression in CR cells.35 Interestingly a recent report showed a reduction in BDNF mRNA in the hippocampus of schizophrenic patients as compared to controls suggesting the presence of decreased trophic support for growth of hippocampal afferents in schizophrenic brain.36 The viral insult in utero may alter BDNF expression with resultant decrease in Reelin production. The phenotypic abnormalities observed by us include global reduction in thickness of neocortex, hippocampus and brain hemisphere (Table 2). Moreover, laminar width is also decreased in cerebral cortex of experimental brains. We also observed decreased nuclear size and increased cell density in cerebral cortical pyramidal cells in the infected brains. The phenotypes in reeler, scrambler and yotari mutations consist of disorganization in the lamination pattern of cerebral and hippocampal cortices37,38 presumably due to absence of Reelin20 and aberrant splicing of mouse disabled I (mdab 1).39–41 However, some similarities do exist between the cerebral cortex phenotype of infected animals and that of the reeler, scrambler and yotari brains, ie, absence of layer I and presence of more pyramidal cells at more superficial locations in the latter phenotypes20,37,39–42 vs decreased thickness of layer I and increased density of pyramidal cells in layers II– VI of infected brains. Despite these similarities, we have not identified specific reeler-like laminar abnormalities and heterotopias in the infected brains. Thus, absence or reduction in Reelin alone can not be responsible for all of the morphologic abnormalities cited previously. Indeed overproduction of BDNF35 or Neurotrophin-443 and Cdk5 mutations44,45 may cause heterotopias and

disorganization of cerebral cortex architectonics. More interestingly, however, are the similarities between brain structure and Reelin production in schizophrenia8,24,25 and what is observed in the infected brains. Impagnatiello and coworkers24,25 have shown reductions of 40–50% in reelin mRNA in neocortex, hippocampus and cerebellum of schizophrenic brains. Selemon et al8 have reported on increased cerebral cortical pyramidal cell density in prefrontal and occipital cortices of schizophrenic brains. These authors reported on decreased somal size of pyramidal cells, and decreased laminar thickness of cerebral cortex in schizophrenic brain suggesting the presence of brain atrophy.8 These last findings are of considerable importance in studying the etiology of schizophrenia and its potential linkage to neurodevelopmental events which may cause abnormal corticogenesis in this disorder.4,5,8 Additionally, a large body of epidemiologic information9,10 links second trimester prenatal human influenza infection and subsequent rise in schizophrenic births. Thus, decrease in Reelin immunoreactive cell counts seen in the brains of infected neonates and reduction in reelin mRNA in brains of schizophrenics24,25 may point to involvement of human influenza viral infection as a potential trigger for abnormal corticogenesis in some forms of schizophrenia. Moreover, decreased expression in reelin mRNA in schizophrenic brains may also be due to DNA polymorphisms in reelin gene, in some schizophrenic family pedigrees.24,25 Several factors need to be considered which may explain the occurrence of observed findings in prenatally infected brains. The virus employed in the present study is a neurotropic H1N1 human influenza virus derived from the original strain, responsible for the 1918 worldwide epidemic responsible for postencephalitic Parkinsonism and psychosis.46 Immunization of rabbits with certain H1N1 influenza viruses including the neurotropic strain NWS/33 used in this study, resulted in the production of autoantibodies to a brain-specific protein of 37 kDa present in neuronal cell bodies of the dentate gyrus, hippocampus, cerebral cortex and cerebellum.47 Presence of infection was not required for induction of these antibodies, since the isolated hemaglutinin of A/Bellamy/42 strain and formaldehyde-fixed WSN virus were essential for induction of this antibody.47 This may explain why viral cytopathic activity or viral specific antigenicity could not be demonstrated in the brains of neonates prenatally treated with HI viral infection (Fatemi et al, unpublished observations). Another potential means of causing brain abnormality may be due to the transfer of maternal antibodies against HI which may recognize a shared epitope between fetal brain and the HI.48 An alternative mechanism of indirect brain injury may involve the production of cytokines and growth factors eg BDNF or activation of its receptor trkB, due to endotoxin or virally-induced pathways in the infected neonates.35,49 Finally, epidemiologic reports also point to terato-


genicity of fever during and following influenza infection.50 Finally, our data are supported by several reports that lesioning of CR cells51–53 may prevent the growth and development of entorhinohippocampal afferent fibers; this could potentially lead to decrease in neuropil as seen in the reduction of thickness in infected neocortex and hippocampus. In summary, we have provided evidence that prenatal viral infection in utero causes global reductions in Reelin expression by Cajal–Retzius cells and other Reelin-positive neurons in cortex and hippocampus of neonatal mice. This effect is evident in all layers of developing cortex and hippocampus and may be potentially responsible for some of the morphologic abnormalities observed in infected brains (Figure 4). These include decreased area measurements in cerebral cortex, hippocampus and total unilateral brain hemisphere (Table 2) as well as abnormal organization of cerebral cortical layers I–VI. Decrease in Reelin production due to HI, either directly or indirectly, may be one of several factors that may cause this abnormal cerebral cortical organization. This and previous reports12,13 provide evidence for deleterious viral effect on growing brains. This study further supports the epidemiologic data linking second trimester viral infection and subsequent increase in schizophrenic births. Acknowledgements Supported by the National Alliance for research on schizophrenia and depression (Young and established Phyllis and Perry Schwartz investigator awards) (SHF); by University of Minnesota Faculty seed grant (SHF), Minnesota Medical Foundation (SHF), Stanley Foundation (SHF) and NIH contract NO1-AI-65291 (RWS). K Nakajima was supported by the Human Frontier Science Program and the Ministry of Education, Science, and Culture of Japan. Care of experimental animals was in accordance with Institutional guidelines. Some of the research data were presented at the 4th Symposium on the Neurovirology and Neuroimmunology of Schizophrenia and Bipolar Disorder, held November 4, 1998 in Bethesda, Maryland and at the Twelfth International Conference on Antiviral Research, held December 5, 1998, in Hawaii. The authors acknowledge the enthusiastic support provided by Dr P Clayton during the course of this study. We thank Drs P Faris, B Hartmann and DK Waid for their critical reviews of this manuscript. The secretarial assistance of Ms Melanie Julian and Ms Amy Stevens is appreciated.

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