Abstract
Schizophrenia is a debilitating chronic mental disorder with prominent frontal and hippocampal dysfunction. While the exact cause of schizophrenia is poorly understood, a number of factors, including neurodevelopmental disruption, have been linked to the aetiology of the disease. Maternal immune activation (MIA) is a neurodevelopmental model of schizophrenia based on epidemiological evidence that prenatal exposure to infections increases the risk of schizophrenia in adulthood. This model often uses a single systemic administration of the synthetic cytokine inducer polyinosinic:polycytidilic acid (polyI:C) during mid-gestation (e.g., gestation day 15) to induce MIA in pregnant rats. A number of anatomical, neurochemical and behavioral features of schizophrenia are evident in the adult MIA rat offspring.
Accumulating evidence implicates altered metabolism of L-arginine, an amino acid with a number of bioactive metabolites, and microglial dysfunction in the pathogenesis of schizophrenia. Recent research has shown altered brain arginine metabolic profile and microglial dysfunction in the adult, as well as adolescent, MIA rat offspring. However, does a single MIA insult during the early stage of neurodevelopment affect brain arginine metabolism and microglia has not been addressed previously. We hypothesized that MIA during neonatal neurodevelopment might alter brain arginine metabolism and affect microglial maturation.
To test these hypotheses, we designed the following three experiments to investigate how polyI:C-induced MIA affected brain arginine metabolism and microglia in rats at the age of postnatal day (PD) 2. Twenty-four pregnant Sprague-Dawley rats were given a single tail vein injection of polyI:C (4.0 mg/kg; MIA) or saline (control) on gestation day 15 (n = 12/group). Experiment 1 quantified the tissue concentrations of L-arginine and its nine downstream metabolites (L-citrulline, L-ornithine, glutamate, glutamine, GABA, agmatine, putrescine, spermidine and spermine) in the forebrain of male and female control and MIA offspring (n=12/sex/group) at PD2 using high performance liquid chromatographic (HPLC) and liquid chromatography/mass spectrometric (LC/MS) assays. There were no significant differences between the control and MIA groups in both sexes for the ten neurochemical variables measured, suggesting that MIA did not have a global effect on brain arginine metabolism at this age.
Since schizophrenia is associated with prominent frontal and hippocampal dysfunction, Experiment 2 measured tissue concentrations of L-arginine and its nine downstream metabolites in the frontal cortex and hippocampus of male and female control and MIA offspring (n=8/sex/group) at PD2. There were increased levels of L-arginine, glutamate, putrescine, spermidine and spermine and glutamate/GABA ratio, but decreased glutamine/glutamate ratio, in both male and female MIA offspring relative to the controls in a region-specific manner. These results provide the first evidence that a single MIA insult during early neurodevelopment altered arginine metabolism in the frontal cortex and hippocampus at the neonatal stage.
Human genetic studies have identified schizophrenia risk genes encoding neuronal nitric oxide synthase (nNOS), one of the key arginine metabolising enzymes. Experiment 3 compared nNOS immunoreactive profiles and microglial migration and maturation in the brains of control and MIA offspring in both sexes (n = 4/sex/group) at PD 2. Immunohistochemistry revealed markedly increased number of nNOS-positive cells in the somatosensory cortex, striatum and the CA3 and dentate gyrus sub-regions of the hippocampus, but not frontal cortex, in the MIA offspring relative to the controls in both sexes. Moreover, there were delayed microglial migration and maturation in both male and female MIA offspring when compared with the controls.
This study, for the first time, demonstrates that a single MIA insult alters the brain arginine metabolism and leads to the up-regulation of nNOS and abnormal development of microglia at the age of PD 2. These findings further support the involvement of L-arginine metabolism and microglia in the pathogenesis of schizophrenia. The changes during early neonatal neurodevelopment may contribute to neuronal and behavioral dysfunctions observed in the juvenile and adult MIA rat offspring.