The interaction between maternal immune activation and alpha 7 nicotinic acetylcholine receptor in regulating behaviors in the offspring

Abstract

Mutation of human chromosome 15q13.3 increases the risk for autism and schizophrenia. One of the noteworthy genes in 15q13.3 is CHRNA7, which encodes the nicotinic acetylcholine receptor alpha 7 subunit (α7nAChR) associated with schizophrenia in clinical studies and rodent models. This study investigates the role of α7nAChR in maternal immune activation (MIA) mice model, a murine model of environmental risk factor for autism and schizophrenia. We provided choline, a selective α7nAChR agonist among its several developmental roles, in the diet of C57BL/6N wild-type dams throughout the gestation and lactation period and induced MIA at mid-gestation. The adult offspring behavior and gene expression profile in the maternal-placental-fetal axis at mid-gestation were investigated. We found that choline supplementation prevented several MIA-induced behavioral abnormalities in the wild-type offspring. Pro-inflammatory cytokine interleukin-6 (Il6) and Chrna7 gene expression in the wild-type fetal brain were elevated by poly(I:C) injection and were suppressed by gestational choline supplementation. We further investigated the gene expression level of Il6 in Chrna7 mutant mice. We found that the basal level of Il6 was higher in Chrna7 mutant fetal brain, which suggests that α7nAChR may serve an anti-inflammatory role in the fetal brain during development. Lastly, we induced MIA in Chrna7(+/-) offspring. The Chrna7(+/-) offspring were more vulnerable to MIA, with increased behavioral abnormalities. Our study shows that α7nAChR modulates inflammatory response affecting the fetal brain and demonstrates its effects on offspring behavior development after MIA.

Keywords: Autism; Choline supplementation; Interleukin-6 (IL-6); Maternal immune activation (MIA); Maternal infection; Nicotinic acetylcholine receptor alpha 7 subunit (α7AChR, CHRNA7, Chrna7); Poly(I:C); Schizophrenia.

Fig. 1

Maternal choline supplementation ameliorated autistic- and schizophrenia-like behaviors in MIA wild-type offspring. (A–B) Maternal choline supplementation prevented the MIA-induced open field anxiety phenotype as assayed by center entries (A) but not center duration (B). (C) Maternal choline supplementation prevented repetitive/compulsive behavior in the marble burying test that was induced by MIA. MIA offspring given the control diet buried significantly more marbles than saline offspring. The MIA-induced marble burying behavior was reduced by maternal choline supplementation. (D–E) Maternal choline supplementation did not prevent the MIA-induced PPI deficit. Saline-Control diet: n = 6 litters; Poly(I:C)-Control diet: n = 7 litters; Saline-Choline diet: n = 7 litters; Poly(I:C)-Choline diet: n = 8 litters. Data are presented as Mean ± SEM. Significant difference between groups is labeled as * p < 0.05, ** p < 0.01, *** p < 0.001. n.s. : not significant.

Fig. 2

α7nAChR expression in the hippocampus of adult MIA offspring. (A) Compared to control offspring, hippocampal Chrna7 mRNA expression was higher in MIA offspring. The elevation of Chrna7 mRNA expression was not affected by maternal choline supplementation diet, however. Chrna7 mRNA expression was normalized by Gapdh. Saline n = 4; Poly(I:C) n = 12, Poly(I:C)+Choline n = 8. (B) Film images of ^125Iα-bungarotoxin binding to transverse sections through the hippocampus of saline and MIA offspring given a control or choline supplementation diet. The dashed lines illustrate how the subdivisions of the hippocampus were delineated. (C–E) The density of ^125Iα–bungarotoxin (α–BTX) binding to sections of hippocampal formation from saline and MIA offspring given control or choline supplementation diets. Generally, no difference was detected among the groups. Male, Saline-Control diet: n = 3 litters; Poly(I:C)-Control diet: n = 3 litters; Saline-Choline diet: n = 3 litters; Poly(I:C)-Choline diet: n = 4 litters. Female. Saline-Control diet: n = 2 litters; Poly(I:C)-Control diet: n = 4 litters; Saline-Choline diet: n = 5 litters; Poly(I:C)-Choline diet: n = 4 litters. MIA and maternal choline supplementation did not affect the density of α–BTX binding in (C) CA3, (D) CA1 and (E) dentate gyrus (DG). Chrna7: nicotinic acetylcholine receptor alpha 7 subunit. Data are presented as Mean ± SEM. Significant difference between groups is labeled as * p < 0.05, ** p < 0.01.

Fig. 3

Cytokines and cholinergic signaling genes were altered in maternal spleen and splenic macrophage by maternal poly(I:C) injection, but not by maternal choline supplementation. (A) Il6 and Tnf mRNA were increased while Ache and Adrb2 were decreased in the dam’s spleen 3 hr after maternal poly(I:C) injection. Choline supplementation did not affect these changes in gene expression. Saline n = 3 dams; Poly(I:C) n = 4 dams, Poly(I:C)+Choline n = 4 dams. (B) Consistent with the results from the spleen, Il6 and Tnf mRNA were increased in macrophages after maternal poly(I:C) injection, and choline supplementation had little effect. Gene expression was normalized by β-actin. Saline n = 3; Poly(I:C) n = 4, Poly(I:C)+Choline n = 4. Ache: acetylcholinesterase, Adrb2: beta-2 adrenergic receptor, Chrna7: nicotinic acetylcholine receptor alpha 7 subunit, Il6: interleukin-6, Tnf: tumor necrosis factor alpha. Data are presented as Mean ± SEM. Significant difference between groups is labeled as * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 4

Cytokines and cholinergic signaling genes were altered in placenta by maternal poly(I:C) injection and maternal choline supplementation. Maternal poly(I:C) injection increased Il6 mRNA in the placenta, and maternal choline supplementation did not prevent this induction. Maternal choline supplementation lowered Chrna7 and Ache mRNA in placenta after poly(I:C) injection. Gene expression was normalized by β-actin. Saline n = 3 litters; Poly(I:C) n = 4 litters, Poly(I:C)+Choline n = 3 litters. Ache: acetylcholinesterase, Chrna7: nicotinic acetylcholine receptor alpha 7 subunit, Il6: interleukin-6. Data are presented as Mean ± SEM. Significant difference between groups is labeled as * p < 0.05, ** p < 0.01.

Fig. 5

Cytokines and cholinergic signaling genes were altered in fetal brain by maternal poly(I:C) injection and maternal choline supplementation. (A) Maternal poly(I:C) injection increased Il6 and Chrna7 mRNA in fetal brain, and maternal choline supplementation prevented these increases. No changes were found in Ache and Slc5a7 among groups. Gene expression was normalized by β-actin. Saline n = 3 litters; Poly(I:C) n = 3–4 litters, Poly(I:C)+Choline n = 3–4 litters. (B) Maternal choline supplementation was associated with increased trend of choline levels in MIA fetal brain. Each n = 3 litters. Ache: acetylcholinesterase, Chrna7: nicotinic acetylcholine receptor alpha 7 subunit, Il6: interleukin-6, Slc5a7: choline transporter 1. Data are presented as Mean ± SEM. Significant difference between groups is labeled as * p < 0.05.

Fig. 6

MIA increased α7nAChR in fetal brain neurons. MIA E12.5 fetal hindbrain displayed more α7nAChR immunostaining than control hindbrain 3 hours following maternal poly(I:C) injection. In hindbrain subregions (A) PPH and (B) PH, α7nAChR was increased in MIA offspring. (C) Optical density quantitation revealed the increased α7nAChR immunostaining in MIA fetal hindbrain. PPH: Saline: n = 3 litters, Poly(I:C): n = 3 litters; PH: Saline: n = 3 litters, Poly(I:C): n = 4 litters. (D) Confocal images demonstrated α7nAChR-positive cells were double-labeled with mature neuronal marker MAP2 (left panel) and the immature neuronal marker DCX (middle panel), but not with the neural stem cell marker nestin (right panel). White arrows indicate the colocalization between α7nAChR and the other markers. PPH: prepontine hindbrain, PH: pontine hindbrain, MAP2: microtubule-Associated Protein 2, DCX: doublecortin. Data are presented as mean ± SEM. Significant difference between groups is labeled as * p < 0.05.

Fig. 7

Decreased Chrna7 in fetal brain led to the elevation of Il6 expression. (A) MIA increased Chrna7 mRNA level in wild-type fetal brain, but not in Chrna7^+/− and Chrna7^−/− fetal brain. (B) MIA increased Il6 mRNA in wild-type fetal brains but not in Chrna7^+/− and Chrna7^−/− fetal brains. Baseline Il6 levels were elevated in Chrna7^+/− and Chrna7^−/− fetal brains. Gene expression was normalized by β-actin.Saline +/+ n = 3 litters, +/− n = 3 litters, −/− n = 3 litters; Poly(I:C) +/+ n = 3 litters, +/− n = 3 litters, −/− n = 2 litters. Chrna7: nicotinic acetylcholine receptor alpha 7 subunit, Il6: interleukin-6. Data are presented as Mean ± SEM. Significant difference between groups is labeled as * p < 0.05, ** p < 0.01, **** p < 0.0001.

Fig. 8

Compared to wild type, Chrna7^+/− offspring were more susceptible to MIA, generally exhibiting more autistic- and schizophrenia-like behaviors. (A–B) Chrna7^+/− MIA offspring displayed anxiety-like behavior in the open field, while wild-type MIA offspring did not. (C) Wild-type MIA offspring displayed more repetitive behavior in the marble burying test compared to saline offspring. The baseline of repetitive behavior in the Chrna7^+/− offspring was higher than in the wild-type saline offspring and there was no effect of MIA in the Chrna7^+/− offspring. (D–E) Chrna7^+/− MIA offspring displayed a PPI deficit at PPI5 and PPI15, while wild-type MIA offspring exhibited normal PPI5 and PPI15. Saline: n = 4–5 litters, Poly(I:C): n = 4–6 litters. Data are presented as Mean ± SEM. Significant difference between groups is labeled as * p < 0.05, ** p < 0.01, *** p < 0.001.