Pharmacological activation of group-II metabotropic glutamate receptors corrects a schizophrenia-like phenotype induced by prenatal stress in mice

Abstract

Prenatal exposure to restraint stress causes long-lasting changes in neuroplasticity that likely reflect pathological modifications triggered by early-life stress. We found that the offspring of dams exposed to repeated episodes of restraint stress during pregnancy (here named 'prenatal restraint stress mice' or 'PRS mice') developed a schizophrenia-like phenotype, characterized by a decreased expression of brain-derived neurotrophic factor and glutamic acid decarboxylase 67, an increased expression of type-1 DNA methyl transferase (DNMT1) in the frontal cortex, and a deficit in social interaction, locomotor activity, and prepulse inhibition. PRS mice also showed a marked decrease in metabotropic glutamate 2 (mGlu2) and mGlu3 receptor mRNA and protein levels in the frontal cortex, which was manifested at birth and persisted in adult life. This decrease was associated with an increased binding of DNMT1 to CpG-rich regions of mGlu2 and mGlu3 receptor promoters and an increased binding of MeCP2 to the mGlu2 receptor promoter. Systemic treatment with the selective mGlu2/3 receptor agonist LY379268 (0.5 mg/kg, i.p., twice daily for 5 days), corrected all the biochemical and behavioral abnormalities shown in PRS mice. Our data show for the first time that PRS induces a schizophrenia-like phenotype in mice, and suggest that epigenetic changes in mGlu2 and mGlu3 receptors lie at the core of the pathological programming induced by early-life stress.

Figure 1

PRS causes an early and long-lasting reduction in the expression of group-II mGlu receptors in the mouse frontal cortex. mGlu2 and mGlu3 receptors mRNA levels in the frontal cortex (FC) of control (Ctrl) and PRS mice at postnatal day (PND) 1 (at birth), 21, and 60 are shown in (a). β-Actin and GPDH were utilized as internal control. Immunoblot analysis of mGlu2/3 receptors is shown in (b). The representative immunoblot shows the band at 200 kDa corresponding to mGlu2/3 receptor dimers. All values (a, b) are means±SEM of six mice. ^*p<0.05. (Student's t-test) vs the corresponding Ctrl value.

Figure 2

PRS causes an increased expression of DNMT1 and an increased amount of DNMT1 bound to the mGlu2 and mGlu3 receptor gene promoter in the frontal cortex of PND 60 mice; and an increased MeCP2 bound to the mGlu2 gene promoter reversed by the mGlu2/3 receptors agonist LY379268 (0.5 mg/kg, i.p.). Immunoblot analysis of DNMT1 is shown in (a). The representative immunoblot shows a single band at the expected molecular size of about 190 kDa. Data of DNMT1 binding to specific promoter regions of the mGlu2 and mGlu3 receptor genes are shown in (b). In (c), increase in the binding of MeCP2 to the mGlu2 receptor promoter in the frontal cortex of PND 60 PRS mice. The effect was totally reversed by the administration of LY379268. A representative immunoblot of MeCP2 in PRS and control (Ctrl) mice after repeated saline or LY379268 injections is shown in (d) (n=5 per group). The expression of mGlu2 and mGlu3 mRNA levels is shown in (e). Values are means±SEM of six mice. The mRNA values were normalized by β-actin and GPDH. ^*p<0.05. (Student's t-test) vs the corresponding Ctrl values or (#) vs saline.

Figure 3

PRS causes an early and long-lasting reduction in the expression of GAD67 and BDNF in the frontal cortex. GAD67 and BDNF-IX mRNA levels in the frontal cortex of control (Ctrl) and PRS mice at PND 1, 21, and 60 are shown in (a). Immunoblot analysis of GAD67 is shown in (b). The representative immunoblot shows a single band at the expected molecular size of 67 kDa. Values are means±SEM of six mice. The mRNA values were normalized by β-actin and GPDH. ^*p<0.05. (Student's t-test) vs the corresponding Ctrl values.

Figure 4

Pharmacological activation of mGlu2/3 receptors corrects the biochemical abnormalities in the frontal cortex of PRS mice. PRS mice were treated with saline (Sal) or LY379268 (0.5 mg/kg, i.p., twice per day for 5 days). A representative immunoblot of DNMT1 and GAD67 in PRS mice is shown in (a). Combined densitometric values of DNMT1 and GAD67 in control (Ctrl) and PRS mice after saline or LY379268 treatment (n=6 per group). Levels of MeCP2 binding to GAD67 and BDNF-IX promoters, and the effect of LY379268 are shown in (b). In (b) the BDNF mRNA levels in PRS mice after LY379268 treatment. Values are means±SEM of six mice. The mRNA values were normalized by β-actin and GPDH. In (a), ^*p<0.05 (Student's t-test) vs the respective values of PRS mice treated with Sal. In (b), ^*p<0.05 (One-way ANOVA+Newman–Keuls) vs values obtained in the offspring of unstressed dams (Ctrl) naïve or saline-treated mice (^*) or vs values obtained in PRS mice treated with LY379268 (#). Values are means±SEM of six mice.

Figure 5

PRS causes schizophrenia-like behavioral abnormalities reversed by treatment with LY379268 in mice. Data of social interaction, locomotor activity, and prepulse inhibition (PPI) of the startle reflex are shown in (a), (b), and (c), respectively. Values were means±SEM of 8–10 mice. ^*p<0.05 (One-way ANOVA+Newman–Keuls) vs the respective groups of mice treated with saline (Sal) (^*), vs the respective control (Ctrl) mice (^**), or vs Ctrl mice treated with LY379268 (#).