Article Figures & Data
Figures
- Figure 1.
Middle but not late prenatal immune challenge suppresses explorative behavior in adulthood. a, Adult mice born to PolyI:C-exposed mothers on GD9 showed a significant deficit in exploratory behavior compared with control offspring, as evident by the reduction in the frequency to enter the central area in the open field. This effect was not observed in animals born to PolyI:C-treated mothers on GD17. ∗p < 0.05, statistical significance based on Fisher's LSD post hoc comparison. b, No group difference in basal locomotor activity was revealed, therefore the suppression of spatial exploration in GD9-PolyI:C offspring cannot be attributed to any changes in general locomotor activity. c, The three panels illustrate the representative pattern of spatial exploration of the three groups in the entire open field (top row) and in the central area (bottom row). n = 10 (5 males and 5 females) in each treatment group. All values are mean ± SEM. Cntr, Control.
- Figure 2.
Emergence of perseverative behavior after late but not mid pregnancy immune challenge. Neither GD9-PolyI:C nor GD17-PolyI:C treatment affected the acquisition of a left–right discrimination task, as evident by both the measures of error to criterion (a) and percentage correct (b) on successive training days. A clear selective deficit in the GD17-PolyI:C group emerged in the subsequent reversal training, indicating a perseverative phenotype. A reversal effect was evident in all groups as suggested by the below-chance performance (represented by the dashed line) on the first day of reversal learning (b). The reversal effect persisted into the second day of reversal training in the GD17-PolyI:C group but not the other treatment conditions. n = 9 (males) in each treatment group. All values are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, statistical significance based on Fisher's LSD post hoc analysis. Cntr, Control.
- Figure 3.
Enhanced sensitivity to inflammation-mediated disturbances in postnatal Reelin expression in the brains of offspring after mid-pregnancy immune challenge. a–c, Distribution of Reelin-immunoreactive cells in the hippocampal formation and dentate gyrus of representative control animals (a) and animals subjected to prenatal PolyI:C exposure on GD9 (b) or GD17 (c). As evident in the images at a higher magnification (indicated by the square in a), prenatal PolyI:C exposure affected Reelin expression particularly in the stratum oriens (sto) of the CA1 subfield. The arrows in b indicate the lack of Reelin-positive cells in the entire stratum oriens of GD9-Polyl:C offspring. e, g, This effect was particularly pronounced GD9-PolyI:C-exposed animals (e) relative to controls (d), leading to a significant reduction in Reelin-positive cells in the brains of GD9-PolyI:C offspring compared with controls (g). f, Prenatal PolyI:C exposure on GD17 only resulted in a marginal reduction in postnatal Reelin immunoreactivity. n = 4 in each treatment group. The values in g are mean ± SEM. ∗p < 0.05, statistical significance based on Fisher's LSD post hoc analysis. Scale bars: a–c, 500 μm; d, e, 50 μm. Cntr, Control; sto, stratum oriens; str, stratum radiatum; pcl, pyramidal cell layer.
- Figure 4.
Reduction in postnatal neurogenesis in the dentate gyrus after prenatal immune challenge. a, c, e, Photomicrographs of coronal brain sections of the hippocampal formation and dentate gyrus were taken from representative control offspring (a) and animals subjected to prenatal PolyI:C exposure on GD9 (c) or GD17 (e) and visualized with immunoperoxidase staining using anti-DCX antibody. b, d, f, As evident in the images at a higher magnification (indicated by the squares in a, c, and e), reduced DXC relative optical density was observed in both the outer granule cell layer and the subgranular cell layer of the dentate gyrus after prenatal PolyI:C exposure, and this effect was primarily independent of prenatal treatment times. The values in g are mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01, statistical significance based on Fisher's LSD post hoc analysis. Scale bars: a, c, e, 500 μm; b, d, f, 50 μm. Cntr, Control; ogcl, outer granule cell layer; gcl, granule cell layer; sgcl, subgranular cell layer.
- Figure 5.
Selective enhancement of postnatal brain apoptosis after immune challenge on late but not middle gestation. a–c, e, Distribution of activated caspase-3-immunoreactive cells in the dorsal dentate gyrus of representative control animals (a) and offspring subjected to prenatal PolyI:C exposure on GD9 (b) or GD17 (c; higher magnification in e). d, An increase in activated caspase-3-positive cells was observed exclusively in animals exposed to prenatal PolyI:C on GD17, whereas the same treatment on GD9 marginally reduced the number of caspase-3-positive cells compared with control brain tissue, leading to a significant difference between the two prenatal PolyI:C conditions. n = 4 in each treatment group. The values in d are mean ± SEM. ∗p < 0.05, statistical significance based on Fisher's LSD post hoc analysis. Scale bars: a–c, 500 μm; e, 50 μm. Cntr, Control.
- Figure 6.
Unaltered postnatal brain gross morphology after prenatal PolyI:C exposure. a, c, e, Photomicrographs of coronal brain sections of the dorsal hippocampal formation were taken from representative control animals (a) and animals subjected to prenatal PolyI:C exposure on GD9 (c) or GD17 (e) and visualized with Nissl/GFAP double staining, which stains neuronal cell bodies and astrocytes, respectively. b, d, f, Note that images on higher magnification (indicated by the squares in a, c, and e) revealed no differences between prenatal PolyI:C-treated [GD9 (d) and GD17 (f)] and control (Cntr; b) offspring in the neuronal cell numbers or in the abundance of pyknotic neurons and astrocytes. Scale bars: a, c, e, 500 μm; b, d, f, 50 μm.
- Figure 7.
Comparison of the maternal serum cytokine responses after immune challenge in middle and late gestation. The maternal administration of PolyI:C (5 mg/kg, i.v.) led to a marked increase in the serum protein levels of IL-1β, IL-6, IL-10, and TNF-α in both gestation day conditions relative to vehicle (saline) treatment, and these effects were generally more pronounced at 3 h after treatment (a) compared with the later sampling time at 6 h after treatment (b). However, whereas the PolyI:C-induced IL-10 and TNF-α responses were stronger in GD9 compared with GD17 dams at the earlier sampling interval, higher serum protein levels of IL-6 and IL-10 were detected in PolyI:C-exposed GD17 dams compared with the PolyI:C-treated GD9 group at 6 h after treatment. The number of pregnant dams in each group was 4, except in the following three groups: GD9-PolyI:C/3 h, n = 6; GD17-PolyI:C/3 h, n = 5; GD17-PolyI:C/6 h, n = 5. The levels of IL-1β in vehicle-treated animals were always below the detection limit (ND), regardless of the gestation day condition, and so were the IL-6 levels in the vehicle-treated GD17 group at both sampling intervals. All values are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, statistical significance based on Fisher's LSD post hoc comparisons between the effects of PolyI:C treatment on GD9 (GD9-PolyI:C vs GD9-saline) and on GD17 (GD17-PolyI:C vs GD17-saline), as well as between the efficacy of PolyI:C treatment at the two treatment days (GD9-PolyI:C vs GD17-PolyI:C) at each sampling interval (3 or 6 h after treatment).
- Figure 8.
Comparison of the fetal brain cytokine responses after immune challenge in middle and late gestation in terms of relative protein levels. The average fetal brain cytokine levels obtained per progeny (n = 3–5 fetuses per progeny) in the two prenatal PolyI:C conditions (GD9 or GD17) are expressed as percentage of deviation from the mean levels obtained after saline treatment conducted on the same gestation day and at the corresponding post-treatment sampling intervals (3 or 6 h). a, Maternal PolyI:C exposure on GD9 reduced the relative IL-10 protein contents in fetal brain tissue compared with vehicle (saline) treatment and led to a massive elevation of the relative fetal brain IL-6 levels at 3 h after treatment. Conversely, the same treatment conducted on GD17 exerted opposite effects on both cytokines at this sampling time. In addition, the relative protein contents of fetal brain IL-1β were only increased after maternal PolyI:C treatment on GD17, as opposed to the marginal reduction observed in GD9-PolyI:C offspring 3 h after treatment. b, At 6 h after treatment, however, a marked elevation IL-1β was detected in the brain of GD9 PolyI:C-exposed offspring, and the relative protein levels of fetal brain IL-6 were increased in PolyI:C-treated offspring independent of treatment times. The number of pregnant dams in each group was 4, except in the following three groups: GD9-PolyI:C/3 h, n = 6; GD17-PolyI:C/3 h, n = 5; GD17-PolyI:C/6 h, n = 5. The levels of TNF-α on GD9 were below detection limits 3 h after treatment. All values are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, statistical significance based on independent Student's t tests between the effects of PolyI:C treatment on GD9 (GD9-PolyI:C vs GD9-saline) and on GD17 (GD17-PolyI:C vs GD17-saline), as well as between the efficacy of PolyI:C treatment at the two treatment days (GD9-PolyI:C vs GD17-PolyI:C) at each sample interval (3 or 6 h).
- Figure 9.
Comparison of the fetal brain cytokine responses after immune challenge in middle and late gestation in terms of relative gene expression levels. The graphs illustrate the fold changes in the relative cytokine gene expression as calculated using the 2-ΔΔCt method; the bars correspond to the minus and plus range of the fold changes. At each sample interval (3 and 6 h), the average fetal brain cytokine levels obtained per progeny (n = 3–4 fetuses per progeny) was submitted to statistical analyses. a, At 3 h after treatment, the relative gene expression levels of IL-1β were reduced in the fetal brains as a result of maternal PolyI:C exposure in both gestation day conditions, whereas a similar reduction in TNF-α expression was only observed in the brains of fetuses derived from immune-challenged GD17 dams. The relative expression levels of IL-6 and IL-10 genes were unaffected by the maternal treatment at this sampling time. b, At 6 h after treatment, maternal immune stimulation on GD17 significantly increased the relative gene expression levels of IL-6, IL-10, and TNF-α, whereas the same treatment conducted on GD9 exerted an opposite effect on the relative IL-10 gene expression levels. The number of pregnant dams in each group was 4, except in the following three groups: GD9-PolyI:C/3 h, n = 6; GD17-PolyI:C/3 h, n = 5; GD17-PolyI:C/6 h, n = 5. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, statistical significance based on independent Student's t tests between the effects of PolyI:C treatment on GD9 (GD9-PolyI:C vs GD9-saline) and on GD17 (GD17-PolyI:C vs GD17-saline), as well as between the efficacy of PolyI:C treatment at the two treatment days (GD9-PolyI:C vs GD17-PolyI:C) at each sample interval (3 or 6 h).
