Defective Corticogenesis and Reduction in Reelin Immunoreactivity in Cortex and Hippocampus of Prenatally Infected Neonatal Mice

Home , Reeler, Reelin

IMMEDIATE COMMUNICATION 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 reelin- positive 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 virus on day 13 of pregnancy. Further evidence from our laboratory showed that prenatal viral infection on Schizophrenia is a severe brain disorder which affects day 9 of pregnancy causes alterations in nNOS and 1% of the world population today.1 Early investigators SNAP-25 levels in day 0 neonatal brains.12,13 We hypo- had suspected a potential biological origin for this dis- thesized that prenatal human influenza viral infec- order.2,3 Recent reports have indicated neuropathologic tion12 on day 9 of pregnancy in C57BL/6 mice may alter and neurochemical abnormalities in postmortem production of Reelin by Cajal–Retzius cells causing brains of patients with schizophrenia.4–9 More specifi- subsequent morphologic or synaptic changes in brains cally, abnormal translocation in NADPH-diaphorase of day 0 neonatal mice. positive cells in frontal and temporal cortices hint at Reelin is a secretory glycoprotein with relative mol- neurodevelopmental causes for schizophrenia.4,5 The ecular mass of about 400 kDa14 and encoded by a long neurodevelopmental etiology of schizophrenia has also mRNA of about 12 kilobases.15,16 The aminoterminus of been supported by recent epidemiologic reports10 Reelin is 25% identical to that of F-spondin,16 a protein showing an association between maternal second trim- secreted by the floor plate of the spinal cord and ester human influenza viral infection and later thought to regulate the adhesion and extension of com- increased risk for development of schizophrenia.10 missural axons17 and to tenascin.18 Reelin RNA is first Despite preponderance of positive epidemiologic data, detectable in the mouse embryonic brain on day 9.5.19 experimental evidence supporting such a claim is lack- It then increases in concentration up to early postnatal ing. A recent report11 showed a mild increase in pyr- days and then declines to adult levels. The first cells amidal cell disarray of dorsal hippocampus in murine producing Reelin are the pioneer Cajal–Retzius neu- neonates born to mothers exposed to human influenza rons 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 Correspondence: SH Fatemi, MD, PhD, University of Minnesota organization of the cortex.16,19,20 The reeler mutant Medical School, Division of Neuroscience Research, Box 392 UMHC, 420 Delaware St SE, Minneapolis, MN 55455, USA. mouse exhibits widespread morphological abnormali- E-mail: fatem002Ȱgold.tc.umn.edu ties in various cortical structures including abnormal Received 30 November 1998; accepted 17 December 1998 positioning of neurons and aberrant orientation of cell Prenatal viral infection and Reelin SH Fatemi et al 146 bodies and fibers.21,22 In the cerebral cortex of the reeler with influenza virus infection). Each lung was also mouse, neurons destined to form the subplate zone weighed to determine if weight gain due to consoli- occupy ectopic positions in superficial cortical layers. dation occurred and then each was homogenized to a Additionally, neurons developed later which are des- 10% (wt/vol) suspension in minimum essential tined to form the cortical plate, fail to bypass pre- medium (MEM) containing Earle’s balanced salt sol- 22 ␮ viously generated neurons. Thus, an inverted pattern ution, 0.1% NaHCO3, 1% sorbitol, and 50 g genta- of cortical development takes place in the mutant mice. micin ml−1. Each lung homogenate was then diluted There is a striking similarity in migration abnormalities through a series of tenfold dilutions and assayed for involving the NADPH-diaphorase positive cells in infectious virus in triplicate in 96-well microplates schizophrenic brains and the reeler phenotype.4,5 The containing a 24-h monolayer of MDCK cells. Virus- human reelin cDNA has been cloned and maps to induced cytopathic effect determined microscopically region 22 on the long arm of chromosome 7.23 Two was used as the infectivity end point. Data were

recent reports showed a 40–50% decrease in reelin expressed as log10 cell culture infectious doses mRNA in brains of schizophrenic patients.24,25 (CCID50) ml−1 by the method of Reed and Muench.26 We investigated the role of human influenza viral By this titration, it was determined that at a dilution (HI) infection prenatally in C57BL/6 mice.12,13 Intra- of 10−5 none of the mice died of the infection, but dis- nasal infection of day 9 pregnant mice with 10−5 played a mean lung consolidation score of 1.4, a mean dilution of HI (H1N1) caused sublethal infection in lung weight of 258 mg (51% higher than normal lungs), pregnant mice.12,13 Day 0 neonatal brain sections from and had a mean virus titer of 105,25 CCID50 ml−1, indi- infected and sham-infected groups were prepared cating that a moderate but sublethal infection had been for immunocytochemistry.12,13 Immunocytochemical induced. This was the virus dose selected for use in localization12,13 of Cajal–Retzius (CR) cells containing the pregnant mouse study. Reelin was accomplished using a specific monoclonal Pregnant mice 9 days after breeding were exposed antibody (CR-50) against mouse reelin.20 Here we show i.n. to a 10−5 dilution of virus by the method described that prenatal viral infection through decrease in pro- below. As controls, additional pregnant mice were duction of Reelin, may cause abnormal neocortical cer- exposed to sterile virus diluent in the same manner as ebral development. 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 Methods and materials group, the mean lung score was 2.0, the mean lung Animals and viral infection weight was 263 mg, and the mean virus titer was 104,4 Female 12–14-week-old specific pathogen-free CCID50 ml−1. In the control group, no lung consoli- C57BL/6 mice were obtained from Simonsen Labora- dation score was seen, the mean lung weight was tories (Gilroy, CA, USA). For initial virus titration stud- 170 mg, and no virus was detected in the lungs. All the ies, the animals were quarantined 24 h prior to use and animals withstood the anesthesia and i.n. procedure in maintained on Wayne Lab Blox and tap water. After a satisfactory manner. being infected, their drinking water contained 0.006% oxytetracycline (Pfizer, New York, NY, USA) to control Immunohistochemistry for possible bacterial infection. In experiments using Pregnant mice were allowed to deliver pups. The day pregnant C57BL/6 mice, the female mice were bred of delivery was considered day 0. Groups of infected with male mice of the approximate same age after at (n = 12 for reelin experiments, n = 8 for calretinin least 1 week of quarantine. Initial pregnancy was experiments, n = 4 for nNOS experiments) and sham- determined by observation of vaginal plug in the ani- infected neonates (n = 9 for reelin experiments, n = 5 mals following breeding. Influenza A/NWS/33 (H1N1) for calretinin experiments, n = 5 for nNOS was obtained from RW Cochran (University of Michi- experiments) were deeply anesthetized. Brains were gan, Ann Arbor). A virus pool was prepared in Maden removed from skull cavities and immersed in phos- Darby canine kidney (MDCK) cells; the virus was phate-buffered 4% paraformaldehyde (pH 7.4) for 7 ampuled and frozen at −80°C until used. Mice were days at 4°C. Coronal sections (10 ␮m) were cut on an anesthetized by intraperitoneal (i.p.) injection of IEC-minitome cryostat (IEC, Needham Heights, MA, approximately 167 mg kg−1 ketamine (Phoenix Scien- USA) and placed on subbed slides. Alternatively, cry- tific, St Joseph, MO, USA), and while under the effects opreserved brains were snap-frozen by immersion in of anesthetic were inoculated by intranasal instillation liquid nitrogen and stored at −85°C for future use. All (i.n.) with 90 ␮l of virus. The virus was diluted 10−4, infected and sham-infected sections of the cortex and 10−4.5,10−5,or10−6 times; these dilutions were based hippocampus were level-matched using the atlas of on previous titrations performed in other mouse spec- embryonic mouse brain.27 Sections were taken from a ies. A total of 10 mice were exposed to each virus minimum of three levels of cortex and hippocampus dilution; occurrence of death was noted daily for 21 in experimental and control mice. Sections were days in five of these animals; the remaining mice were warmed to room temperature (RT), dried, postfixed in killed on infection day 7 and their lungs removed and acetone for 10 min and redried at RT; they were sub- assigned a consolidation score of 0 (normal) to 4 (entire sequently washed three times at RT in phosphate-buff- lung displaying typical plum coloration associated ered saline containing 0.2% Triton X-100 (PBS, 10 mM Prenatal viral infection and Reelin SH Fatemi et al 147 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 PBS- TX-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); gold- conjugated goat anti rabbit IgG against nNOS; gold-conjugated goat anti rabbit IgG against calretinin). Sub- sequently, silver (Goldmark, NJ, USA) enhancement was carried out on all slides, including control sections (without primary antibody) at a dilution of 1:1 of devel- oper 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 termin- ated by washing the slides with distilled water. Slides were then dehydrated through a graded alcohol series and xylene and coverslipped in permount before exam- ination 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 indi- viduals and cells were counted based on immunostain- ing 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 Figure 1 Reelin-positive Cajal–Retzius cells are seen in localized throughout cortical and hippocampal layers cortical layer I and hippocampal marginal zone in prenatally- and counted. Similar large cells resembling CR cells infected (b, d, f, h) and sham-infected brains (a, c, e, g). At ×20 which expressed nNOS and calretinin were also ident- and ×60 magnifications, Cajal–Retzius cells lie horizontally ified and counted in the cortical layer I of control and in layer I of control brain (a and c respectively). At the same infected brains. Moreover cresyl-violet stained CR magnifications, two horizontally located Cajal–Retzius cells are seen (b and d). At ×6.4 and ×40 magnifications, higher cells, pyramidal and nonpyramidal neurons were numbers of Cajal–Retzius cells can be demonstrated in the identified and counted. hippocampus of sham-infected mice (e, g) as compared to The areas were measured using the Micro-bright field infected (f, h) respectively. Stereo Investigator software and included: cortical layer I, cortical layers II–VI and intermediate zone (presumptive white matter), and hippocampal layers were obtained from a minimum of three sections per (marginal zone, stratum radiatum, stratum pyramidale, brain from control (n = 9) and infected (n = 12) mice. and stratum oriens) and subjacent intermediate zone, Mean areas in mm2 ± SD were obtained and subjected and total unilateral brain hemisphere. Measurements to statistical analysis. were made using cresyl violet-stained sections selected Finally, cell density values for reelin-positive CR and from three rostrocaudal areas of the brain approximat- non-CR cells were obtained by dividing the cell count ing to the level of septodorsal hippocampus (level 1), by mean of each area measured from cresyl violet- mid septotemporal hippocampus (level 2) and tempo- stained neighboring sections and expressed as cell roventral hippocampus (level 3). These measurements counts per mm2. Prenatal viral infection and Reelin SH Fatemi et al 148 Statistical analysis (Figure 2 c, d). The other areas of the brains showed Statistical analysis of data was performed using InStat reductions, but these were not statistically significant GraphPad Software (NY, USA) and consisted of the fol- (Figure 2 c, d). When Reelin-positive cell counts for lowing: (1) Analysis of data in Figure 2a, c, d, e and f combined cortical layer I and hippocampal marginal by Bartlett’s test showed that variances were not equal, zone and all other cortical and hippocampal layers thus data were then subjected to Kruskal–Wallis non- were analyzed, statistically significant differences were parametric ANOVA followed by Dunn’s post test to observed in prenatally-infected brains as compared to obtain statistically significant differences between controls (Figure 2 e, for cortical layer I and hippocam- groups; (2) Analysis of Figure 2b data, by Bartlett’s test, pal marginal zone in MST and TV brains, P Ͻ 0.001 showed that variances were equal. Thus one-way and P Ͻ 0.001 respectively; Figure 2f, for other layers ANOVA followed by Tukey–Kramer post test was perin cerebral cortex and the corresponding layers of hip- formed to establish significance; (3) In analysis of data pocampus in MST and TV brains, P Ͻ 0.01 and for Figure 2 g, h, Figure 3 and Table 1, F testing showed P Ͻ 0.001 respectively). Finally, when all Reelin-posi- that variances were not equal. Thus data were sub- tive cell counts were combined according to brain com- jected to Mann–Whitney non-parametric test to estab- partment ie as total of cells in neocortical layer I and lish significance; (4) Finally, analysis of data for Table hippocampal marginal zone (Figure 2g), total of layers 2 used an unpaired t-test to establish significance. II–VI in neocortex and the corresponding layers of hippocampus (Figure 2h), regardless of rostrocaudal position of brain, infected values were statistically signifi- Results cantly reduced (P Ͻ 0.0001 layer I and marginal zone; Quantitation of Reelin-positive cells in cerebral P Ͻ 0.0001 all other layers) as compared to controls. cortex and hippocampus To control for the potentially confounding factor of a Reelin-positive cells were identified and counted in general reduction in tissue mass, an additional set of layer I of the cerebral cortex and the marginal layer of calculations was performed. In this analysis, Reelin- hippocampus and in the cortical and hippocampal lay- positive cell counts were converted to cell density ers and subjacent intermediate zone. Reelin-positive values. Calculation of Reelin-positive cell density cells were observed in both prenatally-infected and values per mm2 showed statistically significant sham-infected neonatal brains (Figure 1). In layer I of reductions in hippocampal (P Ͻ 0.0007) and neo- sham-infected cerebral cortex, Reelin-positive cells cortical (P Ͻ 0.0008) regions in prenatally infected showed morphologic characteristics of CR cells; these brains when compared to control values (Table 1). Cer- cells appeared larger than pyramidal cells with intra- ebral cortical layer I cell densities were unchanged in cellular staining surrounding an oval or round experimental brains as compared to controls (Table 1). unstained nucleus. CR cells exhibited horizontal exten- This is probably due to a larger and significant decrease sions and were identifiable predominantly in layer I (P Ͻ 0.0001) in cerebral cortical layer I area (39% of cerebral cortex and hippocampal marginal zone in decrease) observed in prenatally-infected brains (Table infected and sham-infected brains. Moreover, smaller 2), as opposed to others measured; thus artifactually Reelin-positive cells, presumably GABA-containing increasing the density values for infected cortical layer neurons and interneurons19,28 could be seen scattered I. Moreover, area measurements of cerebral cortical throughout cerebral cortical layers II–VI, as well as in layers II–VI and intermediate zone and total unilateral the developing hippocampal layers in both infected hemispheres showed statistically significant and sham-infected brains (Figure 1). reductions in infected day 0 brains as compared to con- Quantitative assessment of Reelin-positive cells trols (Figure 4, Table 2).19 The hippocampal values, showed significant differences in the number of these despite a large reduction, were not statistically signifi- cells in several areas of the prenatally-infected neo- cant (Table 2). natal brains as compared to controls (Figure 2 a–f, P Ͻ 0.0001, ANOVA). Prenatally-infected cortical layer Quantitation of Calretinin, nNOS and Reelin-positive I and hippocampal marginal zone showed significant CR cells in layer I of cerebral cortex reductions in Reelin-positive CR cell counts in mid We further investigated whether the reductions in septotemporal (MST) and temporal ventral (TV) brain Reelin-positive cell number and density were due to areas (rostrocaudal positions 2 and 3 respectively) changes in synthesis or degradation of Reelin protein when compared to sham-infected brains (for cortical or cell death. It has been known that CR cells do pro- layer I, MST, P Ͻ 0.01, TV, P Ͻ 0.001; for hippocampal duce a number of other markers both during develop- marginal zone, MST, P Ͻ 0.01 and TV brain P Ͻ 0.05). ment and in adult life.29–32 Calretinin immunoreactive The septodorsal (SD) brain cell counts (rostrocaudal CR cell count in layer I of cortex did not differ between position 1) did not differ significantly between the two experimental and control brains (Figure 3). groups (Figure 2a and 2b). Reelin-positive cell counts Additionally, nNOS immunoreactive cell counts did in cortical layers II–VI, again showed statistically sig- not differ significantly in cerebral cortical layer I of nificant reductions in infected MST and TV brain lev- infected and control brains (Figure 3). Moreover, pre- els (cortical layers II–VI, MST P Ͻ 0.001; TV brains natal viral infection in this study occurred on day 9 of P Ͻ 0.01) and in all hippocampal layers except inter- pregnancy, approximately 0.5–2 days prior to genesis mediate zone of TV brains (hippocampus P Ͻ 0.001) of CR cells on day 9.519 to 1120,32 of pregnancy suggest- Prenatal viral infection and Reelin SH Fatemi et al 149

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 Ͻ 0.01; P Ͻ 0.001) and hippocampal marginal zone (P Ͻ 0.01; P Ͻ 0.05) (a and b respectively) and other infected cortical (P Ͻ 0.001, P Ͻ 0.01) and hippocampal (P Ͻ 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 Ͻ 0.001, P Ͻ 0.001 layer I and marginal zone respectively; P Ͻ 0.01, P Ͻ 0.001 other cortical and hippocampal layers respectively). Finally global reelin-positive cell counts in hemispheric cortical layer I and hippocampal marginal zone (P Ͻ 0.0001) and other layers (P Ͻ 0.0001) also showed statistically significant reductions in the infected mice (g and h). Prenatal viral infection and Reelin SH Fatemi et al 150

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 Ͻ 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 infected brains, but point to abnormal production of expressed as cells per mm2 in prenatally-infected and sham- Reelin in infected neurons potentially due to either infected day 0 neonatal brains decreased synthesis or increased degradation of the Reelin molecule (Figure 3). Animals Cerebral Cerebral Hippocampus cortical layer I cortex (layers (all layers) Abnormal cerebral cortical development in prenatally II–VI) infected mice Area measurements in cerebral cortex, hippocampus Sham-infected 211.1 ± 77.1 74.0 ± 60.8 166.2 ± 79.8 and unilateral brain hemispheres were decreased in the (n = 9) infected neonatal brains (Figure 4, Table 2). Specifi- Infected 212.5 ± 75.5 31.4 ± 21.9* 107.9 ± 51.2** = cally, there were statistically significant decreases in (n 12) cerebral cortical layer I (P Ͻ 0.0001, Х 39% decrease), %⌬ – ↓ 57.5% ↓ 35% in overall cerebral cortical layers II–VI including the Ͻ Х ± intermediate zone (P 0.0015, 27.2% decrease), and Values have been expressed as mean SD. Ͻ Ͻ in total unilateral brain hemisphere (P 0.0001, * P 0.0008 (Mann–Whitney test). Х ** P Ͻ 0.0007 (Mann–Whitney test). 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 ing that prenatal viral infection may not have reduced attain statistical significance when compared to control the number of CR cells since cortical layer I calretinin value (Table 2). and nNOS-counts between the two groups would have A review of infected temporal-ventral brains showed to also be altered. Moreover, cresyl violet-stained CR increase in pyramidal cell density of 47% (P Ͻ 0.031) cell counts in layer I of infected brains did not differ in cerebral cortex (data not shown). Estimation of pyr- significantly when compared to control brains (Fatemi amidal cell nuclear size in infected temporal-ventral et al; unpublished data). Collectively, these data indi- cerebral cortex showed statistically significant decrease cated that cell death may not account totally for of 41% (P Ͻ 0.037) in all cortical layers (data not changes in Reelin-positive cell number and density in shown). Prenatal viral infection and Reelin SH Fatemi et al 151 Table 2 Comparison of area measurements (mm2) between prenatally-infected and sham-infected brain areas

Animals Cerebral cortical Cerebral cortex Hippocampus (all layers) Total unilateral brain layer I (layers II–VI) and 1Z a and 1Z a hemisphere

Control 0.56 ± 0.07 4.44 ± 1.08 0.99 ± 0.33 10.3 ± 1.58 (n = 9) Infected 0.34 ± 0.09* 3.23 ± 1.26** 0.81 ± 0.35 7.57 ± 1.97* (n = 12) %⌬ ↓ 39.3% ↓ 27.2% ↓ 18.1% ↓ 26.5%

Values have been expressed as mean ± SD. * P Ͻ 0.0001 (unpaired t-test). ** P Ͻ 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 Ͻ 0.0001 cortical layer I; P Ͻ 0.0015 cortical layers II–VI and WM; P Ͻ 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. Prenatal viral infection and Reelin SH Fatemi et al 152 Discussion disorganization of cerebral cortex architectonics. More interestingly, however, are the similarities between Prenatal human influenza viral infection in utero on brain structure and Reelin production in schizo- day 9 of pregnancy by a neurotropic strain of HI virus phrenia8,24,25 and what is observed in the infected (H1N1), caused global reductions in the production of brains. Impagnatiello and coworkers24,25 have shown Reelin in cerebral cortex and hippocampus of day 0 reductions of 40–50% in reelin mRNA in neocortex, neonatal brains. Moreover, analysis of brains showed hippocampus and cerebellum of schizophrenic brains. abnormal neuronal cell development and migration in Selemon et al8 have reported on increased cerebral the cerebral cortex of experimental brains. cortical pyramidal cell density in prefrontal and Reelin production in reeler mutant mouse is defec- occipital cortices of schizophrenic brains. These tive.16 There are, however, quantitative differences authors reported on decreased somal size of pyramidal between the types of mutations that affect the reelin cells, and decreased laminar thickness of cerebral cor- gene and its synthetic product Reelin.30 Thus, in the tex in schizophrenic brain suggesting the presence of original reeler mutation, no Reelin product could be brain atrophy.8 These last findings are of considerable identified in cerebral cortex, hippocampus and cerebel- importance in studying the etiology of schizophrenia lum of reeler mutant brains.20,33 Alternatively, a trun- and its potential linkage to neurodevelopmental events cated Reelin protein is produced but not secreted in the which may cause abnormal corticogenesis in this dis- 34 ‘Orleans’ reeler mutation. Thus variable expression of order.4,5,8 Additionally, a large body of epidemiologic Reelin could take place secondary to the nature of a information9,10 links second trimester prenatal human 35 mutation or a biochemical defect. A recent report influenza infection and subsequent rise in schizo- indicated that CR cells of the cerebral cortex express phrenic births. Thus, decrease in Reelin immunoreac- receptors for the neurotrophin brain-derived neuro- tive cell counts seen in the brains of infected neonates tropic factor (BDNF) which causes subsequent decrease and reduction in reelin mRNA in brains of schizo- in production of Reelin during early postnatal develop- phrenics24,25 may point to involvement of human ment. Acute BDNF stimulation of cortical neuron cul- influenza viral infection as a potential trigger for abnor- tures and/or overexpression of BDNF in the brains of mal corticogenesis in some forms of schizophrenia. transgenic mice cause a dose-dependent reduction in 35 Moreover, decreased expression in reelin mRNA in Reelin expression in CR cells. Interestingly a recent schizophrenic brains may also be due to DNA polymor- report showed a reduction in BDNF mRNA in the hip- phisms in reelin gene, in some schizophrenic family pocampus of schizophrenic patients as compared to pedigrees.24,25 controls suggesting the presence of decreased trophic Several factors need to be considered which may support for growth of hippocampal afferents in schizo- explain the occurrence of observed findings in pre- phrenic brain.36 The viral insult in utero may alter natally infected brains. The virus employed in the BDNF expression with resultant decrease in Reelin present study is a neurotropic H1N1 human influenza production. virus derived from the original strain, responsible for The phenotypic abnormalities observed by us the 1918 worldwide epidemic responsible for post- include global reduction in thickness of neocortex, hip- 46 pocampus and brain hemisphere (Table 2). Moreover, encephalitic Parkinsonism and psychosis. Immuni- laminar width is also decreased in cerebral cortex of zation of rabbits with certain H1N1 influenza viruses experimental brains. We also observed decreased including the neurotropic strain NWS/33 used in this nuclear size and increased cell density in cerebral study, resulted in the production of autoantibodies to cortical pyramidal cells in the infected brains. The a brain-specific protein of 37 kDa present in neuronal phenotypes in reeler, scrambler and yotari mutations cell bodies of the dentate gyrus, hippocampus, cerebral 47 consist of disorganization in the lamination pattern of cortex and cerebellum. Presence of infection was not cerebral and hippocampal cortices37,38 presumably due required for induction of these antibodies, since the to absence of Reelin20 and aberrant splicing of mouse isolated hemaglutinin of A/Bellamy/42 strain and for- disabled I (mdab 1).39–41 However, some similarities do maldehyde-fixed WSN virus were essential for induc- 47 exist between the cerebral cortex phenotype of infected tion of this antibody. This may explain why viral animals and that of the reeler, scrambler and yotari cytopathic activity or viral specific antigenicity could brains, ie, absence of layer I and presence of more pyr- not be demonstrated in the brains of neonates pre- amidal cells at more superficial locations in the latter natally treated with HI viral infection (Fatemi et al, phenotypes20,37,39–42 vs decreased thickness of layer I unpublished observations). and increased density of pyramidal cells in layers II– Another potential means of causing brain abnor- VI of infected brains. mality may be due to the transfer of maternal anti- Despite these similarities, we have not identified bodies against HI which may recognize a shared epi- specific reeler-like laminar abnormalities and hetero- tope between fetal brain and the HI.48 An alternative topias in the infected brains. Thus, absence or mechanism of indirect brain injury may involve the reduction in Reelin alone can not be responsible for production of cytokines and growth factors eg BDNF all of the morphologic abnormalities cited previously. or activation of its receptor trkB, due to endotoxin or Indeed overproduction of BDNF35 or Neurotrophin-443 virally-induced pathways in the infected neonates.35,49 and Cdk5 mutations44,45 may cause heterotopias and Finally, epidemiologic reports also point to terato- Prenatal viral infection and Reelin SH Fatemi et al 153 genicity of fever during and following influenza infec- 4 Akbarian S, Bunney Jr WE, Potkin SG, Wigal SB, Hagman JO, Sand- tion.50 man CT et al. Altered distribution of nicotinamide-adenine dinucleotide phosphate-diaphorase cells in frontal lobe of schizo- Finally, our data are supported by several reports phrenics implies disturbances of cortical development. Arch Gen 51–53 that lesioning of CR cells may prevent the growth Psychiatry 1993; 50: 169–177. and development of entorhinohippocampal afferent 5 Akbarian S, Vinuela A, Kim JJ, Potkin SG, Bunney WE, Jones EG. fibers; this could potentially lead to decrease in neuro- Distorted distribution of nicotinamide-adenine dinucleotide phos- pil as seen in the reduction of thickness in infected phate-diaphorase neurons in temporal lobe of schizophrenics implies anomalous cortical development. Arch Gen Psychiatry neocortex and hippocampus. 1993; 50: 178–187. In summary, we have provided evidence that pre- 6 Arnold SE, Hyman BT, van Hoesen GW, Damasio AR. Some cytoar- natal viral infection in utero causes global reductions chitectural abnormalities of the entorhinal cortex in schizophrenia. in Reelin expression by Cajal–Retzius cells and other Arch Gen Psychiatry 1991; 48: 625–632. Reelin-positive neurons in cortex and hippocampus of 7 Jakob H, Beckmann H. Gross and histological abnormalities of the entorhinal cortex in schizophrenia. J Royal Soc Med 1989; 82: neonatal mice. This effect is evident in all layers of 466–469. developing cortex and hippocampus and may be 8 Selemon JD, Rajkowska G, Goldman-Rakic PS. Abnormally high potentially responsible for some of the morphologic neuronal density in the schizophrenic cortex. A morphometric abnormalities observed in infected brains (Figure 4). analysis of prefrontal areas and occipital area 17. Arch Gen Psy- These include decreased area measurements in cer- chiatry 1995; 52: 805–818. 9 Bunney BG, Potkin SG, Bunney WE. Neuropathological studies of ebral cortex, hippocampus and total unilateral brain brain tissue in schizophrenia. J Psychiat Res 1997; 31: 159–173. hemisphere (Table 2) as well as abnormal organization 10 Wright P, Takei N, Rifkins L, Murray RM. Maternal influenza, of cerebral cortical layers I–VI. Decrease in Reelin pro- obstetric complications and schizophrenia. Am J Psychiatry 1995; duction due to HI, either directly or indirectly, may 152: 1714–1720. be one of several factors that may cause this abnormal 11 Cotter D, Takei N, Farrell M, Sham P, Quinn P, Larkin C et al. Does prenatal exposure to influenza in mice induce pyramidal cell cerebral cortical organization. This and previous disarray in the dorsal hippocampus? Schiz Res 1995; 16: 233–241. 12,13 reports provide evidence for deleterious viral effect 12 Fatemi SH, Sidwell R, Kist D, Akhter P, Meltzer HY, Bailey K et al. on growing brains. This study further supports the epi- Differential expression of synaptosome-associated protein 25 KDa demiologic data linking second trimester viral infec- [SNAP-25] in hippocampi of neonatal mice following exposure to tion and subsequent increase in schizophrenic births. human influenza virus in utero. Brain Res 1998; 800: 1–9. 13 Fatemi SH, Sidwell R, Akhter P, Sedgwick J, Thuras P, Bailey K et al. Human influenza viral infection in utero increases nNOS Acknowledgements expression in hippocampi of neonatal mice. Synapse 1998; 29: 84–88. Supported by the National Alliance for research on 14 D’Arcangelo G, Nakajima K, Miyata T, Ogawa M, Mikoshiba K, Cur- schizophrenia and depression (Young and established ran T. Reelin is a secreted glycoprotein recognized by the CR-50 Phyllis and Perry Schwartz investigator awards) (SHF); monoclonal antibody. J Neurosci 1997; 17: 23–31. by University of Minnesota Faculty seed grant (SHF), 15 Bar I, Lambert de Rouvroit C, Royaux I, Krizman DB, Dernancourt C, Ruelle D et al. A YAC contig containing the reeler locus with Minnesota Medical Foundation (SHF), Stanley Foun- preliminary characterization of candidate gene fragments. Geno- dation (SHF) and NIH contract NO1-AI-65291 (RWS). mics 1995; 26: 543–549. K Nakajima was supported by the Human Frontier 16 D’Arcangelo G, Miao GG, Chen S-C, Soares HD, Morgan JI, Curran Science Program and the Ministry of Education, T. A protein related to extracellular matrix proteins deleted in the Science, and Culture of Japan. Care of experimental mouse mutant reeler. Nature 1995; 374: 719–723. 17 Klar A, Baldassare M, Jessell TM. F-Spondin: a gene expressed at animals was in accordance with Institutional guide- high levels in the floor plate encodes a secreted protein that pro- lines. Some of the research data were presented at the motes neural cell adhesion and neurite extension. Cell 1992; 69: 4th Symposium on the Neurovirology and Neuroim- 95–110. munology of Schizophrenia and Bipolar Disorder, held 18 Vrucinic N, Chiquet-Ehrismann RR. Tenascin function and regu- November 4, 1998 in Bethesda, Maryland and at the lation of expression. Symp Soc Exp Biol 1993; 47: 155–162. 19 Ikeda Y, Terashima I. Expression of reelin, the gene responsible for Twelfth International Conference on Antiviral the reeler mutation, in embryonic development and adulthood in Research, held December 5, 1998, in Hawaii. The the mouse. Dev Dyn 1997; 210: 157–172. authors acknowledge the enthusiastic support pro- 20 Ogawa M, Miyata T, Nakajima K, Yagyu K, Seike M, Ikenaka K et vided by Dr P Clayton during the course of this study. al. The reeler gene-associated antigen on Cajal-Retzius neurons is We thank Drs P Faris, B Hartmann and DK Waid for a crucial molecule for laminar organization of cortical neurons. Neuron 1995; 14: 899–912. their critical reviews of this manuscript. The secretarial 21 Falconer DS. Two new mutants, ‘trembler’ and ‘Reeler’, with neuro- assistance of Ms Melanie Julian and Ms Amy Stevens logical actions in the house mouse. J Genetics 1951; 50: 192–201. is appreciated. 22 Goffinet AM. An early developmental defect in the cerebral cortex of the Reeler mouse. Anat Embryol 1979; 157: 205–216. 23 De Silva U, D’Arcangelo G, Braden VY, Chen J, Miao GG, Curran References T et al. The human reelin gene: isolation, sequencing, and mapping 1 American Psychiatric Association. Diagnostic and Statistical Man- on chromosome 7. Genome Res 1997; 7: 157–164. ual of Mental Disorders, 4th edn. American Psychiatric Press: 24 Impagnatiello F, Costa E, Guidotti A. Marked decrease of Reelin Washington DC, 1994, pp 1–886. (RL) mRNA expression in the prefrontal (PF) cortex and hippocam- 2 Kraepelin E. Demential Praecox and Paraphrenia. Livingstone: pus of postmortem (PM) schizophrenic brains. Soc Neurosci Abs Edinburgh, Scotland, 1919, pp 1–331. 1997; 23: 1675. 3 Southard EE. On the topical distribution of cortex lesions and ano- 25 Impagnatiello F, Pisce MG, Uzunov D, Pandy G, Guidotti A, Costa malies, in dementia proecox, with some account of their functional E. Reelin mRNA is significantly decreased (ෂ 50%) in several areas significance. Am J Insanity 1915; 71: 603–671. of schizophrenic brain. FASEB Abs 1998; 925: A1470. Prenatal viral infection and Reelin SH Fatemi et al 154 26 Reed LJ, Muench H. A simple method of estimating fifty percent 41 Ware ML, Fox JW, Gonzalez JL, Davis NM, Lambert de Rouvroit C, end points. Am J Hyg 1938; 27: 493–498. Russo CJ et al. Aberrant splicing of a mouse disabled homolog, 27 Schambra UB, Lauder JM, Silver J. Atlas of the Prenatal Mouse mdab 1, in the scrambler mouse. Neuron 1997; 19: 239–249. Brain. Academic Press: San Diego, CA, 1992, pp 1–327. 42 Gonzalez JL, Russo CJ, Goldowitz D, Sweet HO, Davisson MT, 28 Pesold C, Impagnatiello F, Pisu MG, Uzunov DP Costa E, Guidotti Walsh CA. Birthdate and cell marker analysis of scrambler: a novel A et al. Reelin is preferentially expressed in neurons synthesizing mutation affecting cortical development with a reeler-like pheno- gamma-amniobutyric acid in cortex and hippocampus of adult rats. type. J Neurosci 1997; 17: 9204–9211. Proc Natl Acad Sci 1998; 95: 3221–3226. 43 Brustrom JE, Gray-Swain MR, Osborne PA, Pearlman AL. Neuronal 29 Hirotsune S, Takahara T, Sasaki N, Hirose K, Yoshiki A, Ohashi T heterotopias in the developing cerebral cortex produced by Neuro- et al. The reeler gene encodes a protein with an EGF-like motif trophin-4. Neuron 1996; 18: 505–517. expressed by pioneer neurons. Nat Genetics 1995; 10: 77–83. 44 Ohshima T, Ward JM, Huh C-G, Longenecker G, Pant VHC, Brady 30 Marin-Padilla M. Cajal–Rezius cells and the development of the RO et al. Targeted disruption of the cyclin-dependent kinase 5 gene neocortex. TINS 1998; 21: 64–71. results in abnormal corticogenesis, neuronal pathology and perin- 31 Super H, Soriano E, Uylings HBM. The functions of the preplate atal death. Proc Natl Acad Sci 1996; 93: 11173–11178. in development and evolution of the neocortex and hippocampus. 45 Chae T, Kwon YT, Bronson R, Dikkes P, Li E, Tsai L-H. Mice lack- Brain Res Rev 1998; 27: 40–64. ing p35, a neuronal specific activator of CdK5, display cortical 32 D’Arcangelo G, Curran T. Reeler: new tales on an old mutant lamination defects, seizures and adult lethality. Neuron 1996; 18: mouse. BioEssays 1998; 20: 235–244. 29–42. 33 Nakajima K, Mikoshiba K, Miyata T, Kudo C, Ogawa M. Disruption 46 Gotto H, Kawaoka Y. A novel mechanism for the acquisition of of hippocampal development in vivo by CR-50 mAb against Reelin. virulence by a human influenza A virus. Proc Natl Acad Sci 1998; Proc Natl Acad Sci 1997; 94: 8196–8201. 95: 10224–10228. 34 De Bergeyck V, Nakajima K, Lambert de Roubroit C, Naerhuyzen 47 Laing P, Knight JG, Hill JM, Harris AG, Oxford JS, Webster RG et B, Goffinet AM, Miyata T et al. A truncated Reelin protein is pro- al. Influenza viruses induce autoantibodies to a brain-specific 37- duced but not secreted in the ‘Orleans’ reeler mutation (Reln rl- kDa protein in rabbit. Proc Natl Acad Sci 1989; 86: 1998–2002. crl). Mol Brain Res 1997; 50: 85–90. 48 Ruben FL, Thompson DS. Cord blood lymphocyte in vitro 35 Ringstedt T, Linnarsson S, Wagner J, Lendahl U, Kokaia Z, Arenas responses to influenza A antigens after an epidemic of influenza E et al. BDNF regulates reelin expression and Cajal–Retzius cell A/P ort Chalmers/73 (H3N2). Am J Obstet Gynecol 1981; 141: development in the cerebral cortex. Neuron 1998; 21: 305–315. 36 Brouha AK, Shannon–Weickert C, Hyde TM, Herman MM, Murray 443–447. AM, Bigelow LB et al. Reductions in brain derived neurotrophic 49 Goldowitz D, Cusing RC, Laywell E, D’Arcangelo G, Sheldon M, factor mRNA in the hippocampus of patients with schizophrenia. Sweet HO et al. Cerebellar disorganization characteristic of Reeler Soc Neurosci Abs 1996; 22: 1680. in Scrambler mutant mice despite presence of Reelin. J Neurosci 37 Sweet HO, Bronson RT, Johnson KR, Cook SA, Davisson MT. 1997; 17: 8767–8777. Scrambler, a new neurological mutation of the mouse with abnor- 50 Pleet H, Graham Jr JM, Smith DW. Central nervous system and malities of neuronal migration. Mamm Genome 1996; 7: 798–802. facial defects associated with maternal hyperthermia at four to 14 38 Yoneshima H, Nagata E, Matsumoto M, Yamada M, Nakajima K, weeks’ gestation. Pediatrics 1981; 67: 785–789. Miyata T et al. A novel neurological mutant mouse, yotari, which 51 DelRio JA, Heimrich B, Borrell V, Forster E, Drakew A, Alcantara exhibits reeler-like phenotype but expresses CR-50 antigen-reelin. S et al. A role for Cajal–Retzius cells and reelin in the development Neurosci Res 1997; 29: 217–223. of hippocampal connections. Nature 1997; 385: 70–74. 39 Sheldon M, Rice DS, D’Arcangelo G, Yoneshima H, Nakajima K, 52 DelRio JA, Heimrich B, Super H, Borrell V, Frotscher M, Soriano Mikoshiba K et al. Scrambler and yotari dirupt the disabled gene E. Differential survival of Cajal–Retzius cells in organotypic cul- and produce a reeler-like phenotype in mice. Nature 1997; 389: tures of hippocampus and neocortex. J Neurosci 1996; 16: 6896– 730–733. 6907. 40 Howell BW, Hawkes R, Soriano P, Cooper JA. Neuronal position 53 Super H, Sust PP, Soriano E. Survival of Cajal–Retzius cells after in the developing brain is regulated by mouse disabled-1. Nature cortical lesions in newborn mice: a possible role for Cajal-Retzius 1997; 389: 733–737. cells in brain repair. Dev Brain Res 1997; 98: 9–14.