Prenatal immune activation interacts with genetic Nurr1 deficiency in the development of attentional impairments

Abstract

Prenatal exposure to infection has been linked to increased risk of neurodevelopmental brain disorders, and recent evidence implicates altered dopaminergic development in this association. However, since the relative risk size of prenatal infection appears relatively small with respect to long-term neuropsychiatric outcomes, it is likely that this prenatal insult interacts with other factors in shaping the risk of postnatal brain dysfunctions. In the present study, we show that the neuropathological consequences of prenatal viral-like immune activation are exacerbated in offspring with genetic predisposition to dopaminergic abnormalities induced by mutations in Nurr1, a transcription factor highly essential for normal dopaminergic development. We combined a mouse model of heterozygous genetic deletion of Nurr1 with a model of prenatal immune challenge by the viral mimetic poly(I:C) (polyriboinosinic polyribocytidilic acid). In our gene-environment interaction model, we demonstrate that the combination of the genetic and environmental factors not only exerts additive effects on locomotor hyperactivity and sensorimotor gating deficits, but further produces synergistic effects in the development of impaired attentional shifting and sustained attention. We further demonstrate that the combination of the two factors is necessary to trigger maldevelopment of prefrontal cortical and ventral striatal dopamine systems. Our findings provide evidence for specific gene-environment interactions in the emergence of enduring attentional impairments and neuronal abnormalities pertinent to dopamine-associated brain disorders such as schizophrenia and attention deficit/hyperactivity disorder, and further emphasize a critical role of abnormal dopaminergic development in these etiopathological processes.

Figure 1.

Heterozygous Nurr1 deficiency modulates peripheral maternal cytokine levels at basal conditions and following viral-like immune activation. A, The bar plots show serum levels of the prototypical proinflammatory cytokines IL-6 and TNFα and the antiinflammatory cytokine IL-10 in pregnant wt and Nurr1^+/− mice treated with vehicle [saline (SAL)] or the viral mimetic poly(I:C) (POL) (2 mg/kg, i.v.) on gestation day 17. *p < 0.05 and **p < 0.01, based on post hoc group comparisons. Serum levels of IL-1β were below detection limit (0.3 pg/ml) for each experimental condition. B, The bar plots depict the balance between maternal proinflammatory versus antiinflammatory cytokines in terms of the IL-6/IL-10 and TNFα/IL-10 ratios in SAL- or POL-treated wt and Nurr1^+/− dams. *p < 0.05, based on post hoc group comparisons. N(wt, SAL) = 4, N(wt, POL) = 4, N(Nurr1^+/−, SAL) = 5, N(Nurr1^+/−, POL) = 5. All values are means ± SEM.

Figure 2.

Additive effects between prenatal immune activation and Nurr1 deficiency in the development of spontaneous locomotor hyperactivity. Pregnant wt and Nurr1^+/− mice were treated with the viral mimetic poly(I:C) (POL) (2 mg/kg, i.v.) or vehicle [saline (SAL)] on gestation day 17, and the resulting offspring were tested in early adulthood. A, The line plot depicts the total distance moved in the entire open field area as a function of 5 min bins, and the bar plot shows the mean distance moved across the 1 h test period. ***p < 0.001 and ^§p < 0.01, signifying the main effects of genotype and prenatal treatment, respectively. N = 14 in each experimental group; all values are means ± SEM. B, The line plot represents the distance moved in the center zone of the open field as a function of 5 min bins, and the bar plot depicts the mean center zone distance moved across the 1 h test period. ***p < 0.001 and ^§p < 0.01, signifying the main effects of genotype and prenatal treatment, respectively. N = 14 in each experimental group; all values are means ± SEM. C, Computer-generated path drawings of representative SAL- or POL-exposed wt and Nurr1^+/− offspring in the open-field test.

Figure 3.

Additive effects between prenatal immune activation and Nurr1 deficiency in the development of sensorimotor gating deficiency. Pregnant wt and Nurr1^+/− mice were treated with the viral mimetic poly(I:C) (POL) (2 mg/kg, i.v.) or vehicle [saline (SAL)] on gestation day 17, and the resulting offspring were tested in early adulthood. Sensorimotor gating was assessed by PPI of the acoustic startle reflex. A, The line plot shows percentage PPI as a function of three different pulse intensities (P-100, P-110, and P-120, which correspond to 100, 110, and 120 dB_A) and prepulse intensities (+6, +12, and +18 dB_A above background of 65 dB_A); the bar plot depicts the mean percentage PPI across all prepulse and pulse stimuli used. Percentage PPI was calculated by the following expression: 100% × [1 − (mean reactivity on prepulse-plus-pulse trials/mean reactivity on pulse-alone trials)], for each subject, and at each of the three possible pulse and prepulse intensities. *p < 0.05 and ^§p < 0.05, reflecting the main effects of genotype and prenatal treatment, respectively. N = 14 in each experimental group; all values are means ± SEM. B, The line plot shows startle reactivity (in arbitrary units) to pulse-alone trials as a function of the three different pulse intensities (100, 110, and 120 dB_A), and the bar plot depicts the mean startle reactivity across all pulse stimuli used. *p < 0.05, signifying the significant difference between POL-exposed Nurr1^+/− mice and all other groups. N = 14 in each experimental group; all values are means ± SEM. C, Raw score analysis of PPI: The line plot shows the startle reactivity (in arbitrary units) obtained on pulse-alone and prepulse-plus-pulse trials averaged across all three pulse stimuli used. N = 14 in each experimental group; all values are means ± SEM.

Figure 4.

Synergistic effects between prenatal immune activation and Nurr1 deficiency in the development of attentional impairments. Pregnant wt and Nurr1^+/− mice were treated with the viral mimetic poly(I:C) (POL) (2 mg/kg, i.v.) or vehicle [saline (SAL)] on gestation day 17, and the resulting offspring were tested in early adulthood. A, Persistence of the LI effect during the test of conditioned tone-freezing: The line plots show percentage time freezing as function of 1 min bins for the initial 6 min pretone period and for the subsequent 6 min tone period separately for NPE and PE subjects, and the bar plots depict the corresponding mean freezing response over the entire period of tone presentation. **p < 0.01, signifying the significant main effect of preexposure (i.e., LI effect) in POL-exposed Nurr1^+/− offspring. N(NPE) = 7 and N(PE) = 7 in each experimental group; all values are means ± SEM. B, Sustained visual attention in the two-choice visual discrimination test: The line plot depicts choice accuracy (indexed by the percentage correct responses to the cued magazine) for each of the four stimulus-duration trial types (0.5, 1, 2, and 10 s), and the bar plot shows the choice accuracy at the shortest signal duration (0.5 s) condition. *p < 0.05 and **p < 0.01; N(Wt, SAL) = 7, N(Wt, POL) = 7, N(Nurr1^+/−, SAL) = 6, and N(Nurr1^+/−, POL) = 6. All values are means ± SEM.

Figure 5.

Independent effect of prenatal immune activation on spatial working memory deficits. Pregnant wt and Nurr1^+/− mice were treated with the viral mimetic poly(I:C) (POL) (2 mg/kg, i.v.) or vehicle [saline (SAL)] on gestation day 17, and the resulting offspring were tested in early adulthood. A, Spatial working memory was assessed using a dry maze containing 32 holes arranged in a radial design as schematically illustrated. The working memory task was based on the matching-to-position paradigm, in which the animals were required to learn the novel position of a rewarded hole revealed to them on trial 1 of each day to navigate effectively to the same location (i.e., matching) on the subsequent trial on the same day. B, The line plots show the distance moved and latency to find the rewarded hole in trial 2 relative to trial 1. **p < 0.01 (distance) and ***p < 0.001 (latency), reflecting the significant main effects of trials in offspring subjected to prenatal SAL treatment; ^§p < 0.01 and ^#p < 0.05, reflecting the significant difference between prenatally SAL- and POL-treated offspring in trial 2 with respect to distance moved and latency, respectively. N = 7 in each experimental group; all values are means ± SEM. C, Computer-generated search path of representative SAL- or POL-exposed wt and Nurr1^+/− offspring in trial 1 (T1) and trial 2 (T2) of the working memory test. S, Starting position; R, location of the rewarded hole. Note that there was a delay of ∼1 s between placing the animal in the center of the maze and starting the video tracking system, so that the seemingly discrepant starting positions are attributable to this 1 s delay, during which some animals already moved away from the center.

Figure 6.

Independent effects of prenatal immune activation and genetic Nurr1 deficiency on midbrain dopamine cell numbers. A, Schematic coronal brain sections delineating the ventral midbrain areas investigated with reference to bregma [adapted from The Mouse Brain in Stereotaxic Coordinates by Franklin and Paxinos (2008)]. Dopaminergic midbrain cells were quantified in sections ranging from bregma −2.92 to −3.64 mm. 1, VTA; 2a, SNc; 2b, SNr. B, Stereological estimates of TH-positive cells in the SN and VTA of adult wt and Nurr1^+/− offspring subjected to prenatal poly(I:C) (POL) (2 mg/kg, i.v.) or vehicle [saline (SAL)] on gestation day 17. Stereological estimates in the SN took into account TH-positive cells in both the SNc and SNr. **p < 0.01 and ^§p < 0.05, signifying the main effects of genotype and prenatal treatment, respectively. N = 7 males in each experimental group; all values are means ± SEM. C, Coronal brain sections of representative SAL- or POL-exposed wt and Nurr1^+/− offspring stained with anti-TH antibody. The sections highlight the SN and VTA regions as indicated by the dashed lines. Note the decrease of TH-positive cell bodies in the SN of SAL- or POL-exposed Nurr1^+/− offspring (indicated by the arrowheads), and the reduction of TH-positive cells in the VTA of POL-treated wt or Nurr1^+/− offspring (indicated by the white stars).

Figure 7.

Independent and synergistic effects of prenatal immune activation and genetic Nurr1 deficiency on dopaminergic markers in the striatum. A, Schematic coronal brain sections delineating the striatal areas investigated with reference to bregma [adapted from The Mouse Brain in Stereotaxic Coordinates by Franklin and Paxinos (2008)]. Relative optical densities of dopaminergic markers in the NAc core (1a) and NAc shell (1b) were assessed in sections ranging from bregma +1.60 to +0.98 mm; relative optical densities of dopaminergic markers in the CPu (2) were assessed in sections ranging from bregma +1.34 to +0.14 mm. B, Mean + SEM relative optical density of TH in the CPu, NAc core and NAc shell of adult wt and Nurr1^+/− offspring subjected to prenatal poly(I:C) (POL) (2 mg/kg, i.v.) or vehicle [saline (SAL)] on gestation day 17. **p < 0.01, signifying the main effect of genotype. N = 7 males in each experimental group. C, Mean + SEM relative optical density of D₂R in the CPu, NAc core, and NAc shell of SAL- or POL-exposed wt and Nurr1^+/− offspring. *p < 0.05 and **p < 0.01, reflecting the significant difference between POL-exposed Nurr1^+/− offspring and all other groups. N = 7 males in each experimental group. D, Coronal brain sections of representative SAL- or POL-exposed wt and Nurr1^+/− offspring stained with anti-TH antibody. The sections were taken at the level of the ventral striatum highlighting NAc core (1a) and NAc shell (1b) as indicated by the dashed lines. Note the reduction (indicated by the arrowheads) of TH immunoreactivity specifically in the NAc shell region of SAL- or POL-exposed Nurr1^+/− offspring. E, Coronal brain sections of representative SAL- or POL-exposed wt and Nurr1^+/− offspring stained with anti-D₂R antibody. The sections were taken at the level of the ventral striatum highlighting NAc core (1a) and NAc shell (1b) as indicated by the dashed lines. Note the decrease (indicated by the arrowheads) of D₂R immunoreactivity in the NAc core and shell regions emerging selectively in POL-exposed Nurr1^+/− offspring.

Figure 8.

Synergistic effects of genetic Nurr1 deficiency and prenatal immune activation on the prefrontal cortical dopamine system. A, Schematic coronal brain sections delineating the prefrontal cortical areas investigated with reference to bregma [adapted from The Mouse Brain in Stereotaxic Coordinates by Franklin and Paxinos (2008)]. Assessment of the relative optical densities of dopaminergic markers in the mPFC included measurements in the aCG, PrL, and IL cortices and was performed on coronal sections ranging from bregma +2.30 to +1.70 mm. B, Mean + SEM relative optical density of TH in the mPFC of adult wt and Nurr1^+/− offspring prenatally treated with poly(I:C) (POL) (2 mg/kg, i.v.) or vehicle [saline (SAL)] solution on gestation day 17. **p < 0.01; N = 7 males in each experimental group. C, Mean + SEM relative optical density of COMT in the mPFC of SAL- or POL-exposed wt and Nurr1^+/− offspring. *p < 0.05; N = 7 males in each experimental group. D, Coronal brain sections of representative SAL- or POL-exposed wt and Nurr1^+/− offspring stained with anti-TH antibody. The sections were taken at the level of the mPFC highlighting TH-positive fibers in the aCG and PrL cortices. Note the marked decrease (indicated by the arrowheads) of TH-positive fibers emerging selectively in POL-exposed Nurr1^+/− offspring. E, Coronal brain sections of representative SAL- or POL-exposed wt and Nurr1^+/− offspring stained with anti-COMT antibody. The sections were taken at the level of the mPFC highlighting COMT protein expression in the aCG and PrL cortices. The sections were color-coded for the purpose of facilitating the visualization of differential COMT protein expression; strongest staining intensities are shown in yellow, while the background is represented in dark purple (bar inset). fmi, Forceps minor corpus callosum.