A longitudinal examination of the neurodevelopmental impact of prenatal immune activation in mice reveals primary defects in dopaminergic development relevant to schizophrenia

Abstract

Prenatal exposure to infection is a significant environmental risk factor in the development of schizophrenia and related disorders. Recent evidence indicates that disruption of functional and structural dopaminergic development may be at the core of the developmental neuropathology associated with psychosis-related abnormalities induced by prenatal exposure to infection. Using a mouse model of prenatal immune challenge by the viral mimic polyriboinosinic-polyribocytidilic acid, the present study critically evaluated this hypothesis by longitudinally monitoring the effects of maternal immune challenge during pregnancy on structural and functional dopaminergic development in the offspring from fetal to adult stages of life. Our study shows that prenatal immune challenge leads to dopaminergic maldevelopment starting as early as in the fetal stages of life and produces a set of postnatal dopaminergic abnormalities that is dependent on postnatal maturational processes. Furthermore, our longitudinal investigations reveal a striking developmental correspondence between the ontogeny of specific dopaminergic neuropathology and the postnatal onset of distinct forms of dopamine-dependent functional abnormalities implicated in schizophrenia. Prenatal immune activation thus appears to be a significant environmental risk factor for primary defects in normal dopaminergic development and facilitates the expression of postnatal dopamine dysfunctions involved in the precipitation of psychosis-related behavior. Early interventions targeting the developing dopamine system may open new avenues for a successful attenuation or even prevention of psychotic disorders following neurodevelopmental disruption of dopamine functions.

Figure 1.

Age-dependent alterations in midbrain dopamine cell numbers following prenatal immune challenge. Pregnant mice were exposed to the viral mimic Poly-I:C or vehicle treatment, and the effects on midbrain dopamine cell numbers were investigated in the resulting offspring at the fetal (GD19), peripubertal (PND35), and adult (PND70) stages of development using stereological estimations of TH-positive cells on serial coronal brain sections. A, Maternal Poly-I:C treatment significantly increased the number of TH-positive cells in the fetal SN relative to maternal vehicle treatment. At the peripubertal stage of development, prenatally immune challenged and control offspring did not differ in the number of TH-positive neurons in the SN. However, a significant increase in the number of SN TH-positive cells was noticeable in adult offspring born to Poly-I:C-challenged mothers in comparison with age-matched control offspring. *p < 0.05, based on Fisher's LSD post hoc group comparisons of GD19 or PND70 specimen following the presence of a significant two-way interaction in the initial 2 × 3 (prenatal treatment × age) ANOVA (F_(2,63) = 4.94, p < 0.05). B, Prenatal Poly-I:C exposure led to a significant increase in TH-positive cells in the VTA specifically in adult (but not peripubertal or fetal) offspring compared with adult offspring of control mothers. *p < 0.05, based on Fisher's LSD post hoc group comparisons of PND70 specimen following the presence of a significant two-way interaction in the initial 2 × 3 (prenatal treatment × age) ANOVA (F_(2,63) = 3.62, p < 0.05). C, Representative images of coronal brain sections of fetal (GD19) and adult (PND70) offspring derived from vehicle- or Poly-I:C-treated mothers stained for TH protein by immunohistochemistry. The images show TH cell stainings in the fetal SN and VTA, and in the adult SN. For all developmental stages, TH-positive cells in the midbrain were clearly identifiable by the appearance of darkly stained cell bodies. Scale bars, 250 μm. All values in A and B are means ± SEM. The numbers of offspring included in the analyses were N(GD19-vehicle) = 12, N(GD19-Poly-I:C) = 11, N(PND35-vehicle) = 12, N(PND35-Poly-I:C) = 11, N(PND70-vehicle) = 11, N(PND70-Poly-I:C) = 12.

Figure 2.

Age-dependent alterations in mesencephalic expression of the orphan nuclear transcription factor Nurr1 following prenatal immune challenge. Pregnant mice were exposed to the viral mimic Poly-I:C or vehicle treatment, and the effects on Nurr1 protein expression in midbrain structures were investigated in the resulting offspring at the fetal (GD19), peripubertal (PND35), and adult (PND70) stages of development using stereological estimations of Nurr1-positive cells on serial coronal brain sections. A, Maternal Poly-I:C treatment significantly increased the number of Nurr1-positive cells in the fetal SN relative to maternal vehicle treatment. At the peripubertal stage of development, prenatally immune challenged and control offspring did not differ in the number of Nurr1-positive neurons in the SN region. However, a significant increase in the number of SN Nurr1-positive cells was noticeable in adult offspring born to Poly-I:C-challenged mothers in comparison with age-matched control offspring. ***p < 0.001, based on Fisher's LSD post hoc group comparisons of GD19 or PND70 specimen following the presence of a significant two-way interaction in the initial 2 × 3 (prenatal treatment × age) ANOVA (F_(2,63) = 17.38, p < 0.001). B, Prenatal Poly-I:C exposure led to a significant increase in Nurr1-positive cells in the VTA specifically in adult (but not peripubertal or fetal) offspring compared with adult offspring of control mothers. ***p < 0.001, based on Fisher's LSD post hoc group comparisons of PND70 specimen following the presence of a significant two-way interaction in the initial 2 × 3 (prenatal treatment × age) ANOVA (F_(2,63) = 4.06, p < 0.05). C, Representative images of coronal brain sections of fetal (GD19) and adult (PND70) offspring derived from vehicle- or Poly-I:C-treated mothers stained for Nurr1 protein by immunohistochemistry. The images show Nurr1 cell stainings in the fetal SN and VTA, and in the adult SN. For all developmental stages, Nurr1-positive cells in the midbrain were clearly identifiable by the appearance of darkly stained cell bodies. Scale bars, 250 μm. All values in A and B are means ± SEM. The numbers of offspring included in the analyses were N(GD19-vehicle) = 12, N(GD19-Poly-I:C) = 11, N(PND35-vehicle) = 12, N(PND35-Poly-I:C) = 11, N(PND70-vehicle) = 11, N(PND70-Poly-I:C) = 12.

Figure 3.

Age- and region-specific alterations in striatal TH expression following prenatal immune challenge. Pregnant mice were exposed to the viral mimic Poly-I:C or vehicle treatment, and the effects on striatal TH expression were investigated in the resulting offspring at the fetal (GD19), peripubertal (PND35), and adult (PND70) stages of development using optical densitometry of immunohistochemically stained coronal brain sections. A, Maternal Poly-I:C treatment significantly increased TH immunoreactivity in the caudate putamen region of the fetal striatum relative to maternal vehicle treatment. However, at the peripubertal stage of development, offspring born to Poly-I:C-treated mothers displayed a significant reduction in TH immunoreactivity in the CPu relative to age-matched control offspring, but no significant group differences in CPu TH immunoreactivity were present at adult age. *p < 0.05, based on Fisher's LSD post hoc group comparisons of GD19 or PND35 specimen following the presence of a significant two-way interaction in the initial 2 × 3 (prenatal treatment × age) ANOVA (F_(2,63) = 6.11, p < 0.01). B, Prenatal Poly-I:C exposure did not significantly influence TH immunoreactivity in the NAc region of the fetal striatum relative to maternal vehicle treatment. However, the prenatal immunological manipulation significantly decreased TH immunoreactivity in both NAc core and shell at the peripubertal stage of development, but it significantly increased NAc core and shell TH immunoreactivity in adult offspring relative to age-matched control offspring. *p < 0.05 and **p < 0.01, based on Fisher's LSD post hoc group comparisons of PND35 or PND70 specimen following the presence of a significant two-way interaction in the initial 2 × 2 (prenatal treatment × postnatal age) ANOVA (NAc core: F_(1,42) = 14.79, p < 0.001; NAc shell: F_(1,42) = 15.52, p < 0.05). C, Representative images of coronal brain sections of fetal (GD19), peripubertal (PND35), and adult (PND70) offspring derived from vehicle- or Poly-I:C-treated mothers stained for TH by immunohistochemistry. 1, CPu; 2, NAc; 2A, NAc core; 2B, NAc shell. Scale bars, 500 μm. All values in A and B are means ± SEM. The numbers of offspring included in the analyses were N(GD19-vehicle) = 12, N(GD19-Poly-I:C) = 11, N(PND35-vehicle) = 12, N(PND35-Poly-I:C) = 11, N(PND70-vehicle) = 11, N(PND70-Poly-I:C) = 12.

Figure 4.

Age- and region-specific reduction in striatal DAT expression following prenatal immune challenge. Pregnant mice were exposed to the viral mimic Poly-I:C or vehicle treatment, and the effects on striatal DAT expression were investigated in the resulting offspring at the fetal (GD19), peripubertal (PND35), and adult (PND70) stages of development using optical densitometry of immunohistochemically stained coronal brain sections. A, Offspring born to Poly-I:C-treated mothers displayed a significant decrease in DAT immunoreactivity in the CPu specifically at peripubertal age in comparison with age-matched offspring born to vehicle-treated control mothers. **p < 0.01, based on Fisher's LSD post hoc group comparison of PND35 specimen following the presence of a significant two-way interaction in the initial 2 × 3 (prenatal treatment × age) ANOVA (F_(2,63) = 5.60, p < 0.01). B, Prenatal Poly-I:C exposure also led to a significant reduction in DAT immunoreactivity in the fetal NAc region compared with fetuses derived from control mothers. *p < 0.05, based on one-way ANOVA with the between-subjects factor of prenatal treatment ANOVA (F_(1,21) = 10.36, p < 0.01). In addition, peripubertal (but not adult) offspring born to Poly-I:C-treated mothers showed a significant reduction in DAT immunoreactivity in both the NAc core and shell subregions relative to peripubertal control offspring. *p < 0.01, based on Fisher's LSD post hoc group comparison of PND35 specimen following the presence of a significant two-way interaction in the initial 2 × 2 (prenatal treatment × postnatal age) ANOVA (NAc core: F_(1,42) = 4.72, p < 0.05; NAc shell: F_(1,42) = 4.56, p < 0.05). C, Representative images of coronal brain sections of fetal (GD19) and peripubertal (PND35) offspring derived from vehicle- or Poly-I:C-treated mothers stained for DAT by immunohistochemistry. 1, CPu; 2, NAc; 2A, NAc core; 2B, NAc shell. Scale bars, 500 μm. All values in A and B are means ± SEM. The numbers of offspring included in the analyses were N(GD19-vehicle) = 12, N(GD19-Poly-I:C) = 11, N(PND35-vehicle) = 12, N(PND35-Poly-I:C) = 11, N(PND70-vehicle) = 11, N(PND70-Poly-I:C) = 12.

Figure 5.

Postpubertal onset of altered D1R expression in striatal regions following prenatal immune challenge. Pregnant mice were exposed to the viral mimic Poly-I:C or vehicle treatment, and the effects on striatal D1R expression were studied in the resulting offspring at the fetal (GD19), peripubertal (PND35), and adult (PND70) stages of development using optical densitometry of immunohistochemically stained coronal brain sections. A, Offspring born to Poly-I:C-treated mothers displayed a significant increase in D1R immunoreactivity in the CPu specifically at adult age in comparison with adult offspring born to vehicle-treated control mothers. *p < 0.05, based on Fisher's LSD post hoc group comparison of PND70 specimen following the presence of a significant two-way interaction in the initial 2 × 3 (prenatal treatment × age) ANOVA (F_(2,63) = 3.94, p < 0.05). B, Prenatal Poly-I:C exposure also significantly increased D1R immunoreactivity in the NAc shell but not in the core subregion in the adult offspring relative to adult control offspring. *p < 0.05, based on Fisher's post hoc group comparison of PND70 specimen following the presence of a significant two-way interaction in the initial 2 × 2 (prenatal treatment × postnatal age) ANOVA (F_(1,42) = 3.31, p < 0.05). C, Representative images of coronal brain sections of adult (PND70) offspring born to vehicle- or Poly-I:C-treated mothers stained for D1R by immunohistochemistry. 1, CPu; 2A, NAc core; 2B, NAc shell. Scale bar, 500 μm. All values in A and B are means ± SEM. The numbers of offspring included in the analyses were N(GD19-vehicle) = 12, N(GD19-Poly-I:C) = 11, N(PND35-vehicle) = 12, N(PND35-Poly-I:C) = 11, N(PND70-vehicle) = 11, N(PND70-Poly-I:C) = 12.

Figure 6.

Postpubertal onset of altered D2R expression in striatal regions following prenatal immune challenge. Pregnant mice were exposed to the viral mimic Poly-I:C or vehicle treatment, and the effects on striatal D2R expression were studied in the resulting offspring at the fetal (GD19), peripubertal (PND35), and adult (PND70) stages of development using optical densitometry of immunohistochemically stained coronal brain sections. A, Offspring born to Poly-I:C-treated mothers did not display any significant differences in D2R immunoreactivity in the CPu at the fetal, peripubertal, or adult stage of development compared with age-matched offspring born to vehicle-treated control mothers. B, However, prenatal Poly-I:C exposure significantly increased D2R immunoreactivity specifically in the NAc shell but not in the core subregion in the adult offspring relative to adult control offspring. *p < 0.05, based on Fisher's LSD post hoc group comparison of PND70 specimen following the presence of a significant two-way interaction in the initial 2 × 2 (prenatal treatment × postnatal age) ANOVA (F_(1,42) = 4.28, p < 0.05). C, Representative images of coronal brain sections of adult (PND70) offspring born to vehicle- or Poly-I:C-treated mothers stained for D2R by immunohistochemistry. 1, CPu; 2A, NAc core; 2B, NAc shell. Scale bar, 500 μm. All values in A and B are means ± SEM. The numbers of offspring included in the analyses were N(GD19-vehicle) = 12, N(GD19-Poly-I:C) = 11, N(PND35-vehicle) = 12, N(PND35-Poly-I:C) = 11, N(PND70-vehicle) = 11, N(PND70-Poly-I:C) = 12.

Figure 7.

Confirmation of the adult dopaminergic phenotype in perfused adult brain samples. Pregnant mice were exposed to the viral mimic Poly-I:C or vehicle treatment, and immunoreactivities of TH, DAT, D1R, and D2R were assessed in the adult (PND70) offspring according to standard procedures using perfused brain samples. A, Prenatal Poly-I:C exposure led to a significant increase in the number of TH-positive cells in the SN (including both SNc and SNr) and the VTA compared with prenatal vehicle treatment. B, Offspring born to Poly-I:C-treated mothers displayed enhanced TH immunoreactivity specifically in the nucleus accumbens core (NAc core) and shell (NAc shell) subregions (indicated by the white arrows) but not in the CPu. C, Prenatal Poly-I:C exposure led to a significant increase in D1R immunoreactivity in the CPu and NAc core regions of the striatum (indicated by the white arrows) relative to prenatal control treatment. D, Offspring born to Poly-I:C-exposed mothers displayed enhanced D2R immunoreactivity specifically in the NAc core NAc shell subregions (indicated by the white arrows) but not in the CPu. All values are means ± SEM. *p < 0.05, based on independent Student's t tests (two tailed). For all analyses, the numbers of offspring included in the analyses were N(vehicle) = 8, N(Poly-I:C) = 7. 1, CPu; 2A, NAc core; 2B, NAc shell. Scale bars, 250 μm.

Figure 8.

Prenatal immune activation induces enhancement of the behavioral sensitivity to acute amphetamine treatment regardless of the offspring's postnatal age. The behavioral sensitivity of prenatally Poly-I:C- or vehicle-treated offspring to a low dose of AMPH (2.5 mg/kg, i.p.) was assessed in an open field arena when they reached the peripubertal (PND35) or adult (PND70) stage of development. The line plots show locomotor activity as indexed by the total distance traveled in the entire open field across bins of 5 min during the initial acclimatization period as well as following saline (SAL) and AMPH administration; the bar plots represent the mean distance moved across the entire 80 min period following AMPH administration. A, No significant differences in basal locomotor activity (as measured during the initial acclimatization period and following SAL administration) were noticeable between Poly-I:C and control offspring at peripubertal age. Acute treatment with the low dose of AMPH led to a modest increase in locomotor activity in peripubertal offspring born to control mothers. The locomotor-enhancing effects of AMPH were significantly increased in peripubertal offspring born to Poly-I:C-treated mothers. *p < 0.05 represents the significant main effect of prenatal treatment in the 2 × 16 (prenatal treatment × bins) repeated-measures ANOVA of distance moved following AMPH treatment (F_(1,25) = 4.27). The numbers of offspring included were N(vehicle) = 14, N(Poly-I:C) = 13. B, There were also no significant differences in basal locomotor activity between Poly-I:C and control offspring at the adult stage of life. Acute treatment with the low dose of AMPH led to marked increase in locomotor activity in peripubertal offspring born to control mothers. Again, the locomotor-stimulating effects of AMPH were significantly enhanced in adult offspring born to Poly-I:C-treated mothers. *p < 0.05 represents the significant main effect of prenatal treatment in the 2 × 16 (prenatal treatment × bins) repeated-measures ANOVA of distance moved following AMPH treatment (F_(1,20) = 5.36). The numbers of offspring included were N(vehicle) = 10, N(Poly-I:C) = 12. All values in A and B are means ± SEM.

Figure 9.

Postpubertal emergence of altered behavioral sensitivity to acute apomorphine treatment following prenatal immune activation. Locomotor activity was assessed in peripubertal (PND35) and adult (PND70) Poly-I:C and control offspring following acute treatment with a low dose of APO (0.75 mg/kg, s.c.) or vehicle (vitamin C solution, VitC) in a sawdust embedded wire-mesh cylinder. The line plots show horizontal locomotor activity as indexed by the total distance traveled in the test apparatus across bins of 5 min; the bar plots represent the mean distance moved across the entire 60 min period. A, APO treatment similarly depressed horizontal locomotor activity in peripubertal (PND35) Poly-I:C and control offspring relative to corresponding vehicle (VitC) treatment. **p < 0.01 represents the significant main effect of drug treatment in the 2 × 2 × 12 (prenatal treatment × drug treatment × bins) repeated-measures ANOVA of horizontal distance moved (F_(1,29) = 8.82). The numbers of offspring included were N(VitC-treated vehicle offspring) = 7, N(APO-treated vehicle offspring) = 10, N(VitC-treated Poly-I:C offspring) = 7, N(APO-treated Poly-I:C offspring) = 9. B, In contrast to the persistent decrease in locomotor activity revealed in adult control (vehicle) offspring following acute APO treatment, horizontal locomotor activity in APO-treated adult Poly-I:C offspring was characterized by an initial decrease and a subsequent increase. The secondary increase in locomotor activity observed in APO-treated adult Poly-I:C offspring was most pronounced between 15 and 30 min following APO treatment and led to a significant attenuation of the APO-induced depression of locomotor activity typically seen in APO-treated adult control offspring. **p < 0.01, based on Fisher's LSD post hoc group comparisons following the presence of a significant (F_(1,37) = 6.25, p < 0.05) prenatal treatment × drug treatment interaction in the initial 2 × 2 × 12 (prenatal treatment × drug treatment × bins) repeated-measures ANOVA of horizontal distance moved. The numbers of offspring included were N(VitC-treated vehicle offspring) = 9, N(APO-treated vehicle offspring) = 12, N(VitC-treated Poly-I:C offspring) = 8, N(APO-treated Poly-I:C offspring) = 12. All values in A and B are means ± SEM.

Figure 10.

Postpubertal emergence of sensorimotor gating deficits following prenatal immune activation. The effects of prenatal Poly-I:C-induced immune challenge, relative to prenatal vehicle treatment, on sensorimotor gating in peripuberty (PND35) and adulthood (PND70) were investigated using the paradigm of PPI of the acoustic startle reflex. The line plots show %PPI as a function of the three intensities of prepulse (71, 77, and 83 dB_A, which corresponded to 6, 12, and 18 dB_A above background white noise, respectively) and of the three intensities of pulse (P-100, P-110, and P-120, which corresponded to pulse intensities of 100, 110, and 120 dB_A, respectively). The bar plots depict mean %PPI across all prepulse and pulse levels used. A, No significant differences in PPI were apparent between peripubertal offspring born to Poly-I:C- and vehicle-treated mothers. The numbers of offspring included were N(vehicle) = 14, N(Poly-I:C) = 14. B, However, a significant overall reduction in %PPI emerged in prenatally Poly-I:C-treated offspring at adult age relative to adult control offspring. The PPI-disrupting effects of prenatal Poly-I:C exposure seemed most pronounced in conditions, in which the highest pulse stimulus (P-120) was used. **p < 0.01 represents the significant main effect of prenatal treatment in the 2 × 3 × 3 (prenatal treatment × prepulse level × pulse level) ANOVA of %PPI (F_(1,26) = 11.01). The numbers of offspring included were N(vehicle) = 15, N(Poly-I:C) = 13. All values in A and B are means ± SEM.

Figure 11.

Effects of pharmacological dopamine D1 or D2 receptor blockade on prepulse inhibition and prepulse-induced startle reactivity in adult offspring born to immune-challenged or control mothers. The line plots show %PPI as a function of the three intensities of prepulse (71, 77, and 83 dB_A, which corresponded to 6, 12, and 18 dB_A above background white noise, respectively) and of the three intensities of pulse (P-100, P-110, and P-120, which corresponded to pulse intensities of 100, 110, and 120 dB_A, respectively). The bar plots present mean %PPI across all prepulse and pulse levels used. A, The prenatal Poly-I:C-induced impairment in PPI was normalized by acute administration of the preferential dopamine D1 receptor antagonist SCH23390 (1 mg/kg, s.c.) or by the preferential dopamine D2 receptor antagonist raclopride (3 mg/kg, i.p.). *p < 0.05, based on Fisher's LSD post hoc group comparisons following the presence of a significant (F_(2,55) = 4.55, p < 0.05) prenatal treatment × drug treatment interaction in the initial 2 × 3 × 3 × 3 (prenatal treatment × drug treatment × prepulse level × pulse level) ANOVA. B, Vehicle-treated offspring born to Poly-I:C-exposed mothers displayed a significant enhancement in prepulse-induced startle reactivity [in arbitrary units (AU)] compared with vehicle-treated offspring born to control mothers. This effect of the prenatal Poly-I:C challenge was normalized by acute administration of SCH23390 (1 mg/kg, s.c.) or raclopride (3 mg/kg, i.p.). On the other hand, acute raclopride treatment significantly increased prepulse-induced startle reactivity in offspring born to control mothers. *p < 0.05 and **p < 0.01, based on Fisher's LSD post hoc group comparisons following the presence of a significant (F_(2,177) = 19.73, p < 0.001) prenatal treatment × drug treatment interaction in the initial 2 × 3 × 3 (prenatal treatment × drug treatment × prepulse level) ANOVA. The numbers of offspring included were N(control offspring/vehicle) = 10, N(control offspring/SCH23390) = 10, N(control offspring/raclopride) = 10, N(Poly-I:C offspring/vehicle) = 11, N(Poly-I:C offspring/SCH23390) = 10, N(Poly-I:C offspring/raclopride) = 10. All values in A and B are means ± SEM.