Glutathione deficit during development induces anomalies in the rat anterior cingulate GABAergic neurons: Relevance to schizophrenia

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Abstract

A series of studies in schizophrenic patients report a decrease of glutathione (GSH) in prefrontal cortex (PFC) and cerebrospinal fluid, a decrease in mRNA levels for two GSH synthesizing enzymes and a deficit in parvalbumin (PV) expression in a subclass of GABA neurons in PFC. GSH is an important redox regulator, and its deficit could be responsible for cortical anomalies, particularly in regions rich in dopamine innervation. We tested in an animal model if redox imbalance (GSH deficit and excess extracellular dopamine) during postnatal development would affect PV-expressing neurons. Three populations of interneurons immunolabeled for calcium-binding proteins were analyzed quantitatively in 16-day-old rat brain sections. Treated rats showed specific reduction in parvalbumin immunoreactivity in the anterior cingulate cortex, but not for calbindin and calretinin. These results provide experimental evidence for the critical role of redox regulation in cortical development and validate this animal model used in schizophrenia research.

Introduction

There is increasing evidence indicative for alterations in the circuitry of the prefrontal cortex (PFC) that uses the γ-amino butyric acid (GABA) as a neurotransmitter in schizophrenia. A significant decrease in the brain tissue content of GABA, decreased density of GABA membrane transporter-1 (GAT-1) immunoreactive (IR) axon cartridges (Woo et al., 1998) and levels of GAT-1 mRNA (Ohnuma et al., 1999), and a reduction in numbers of interneurons expressing mRNA for the 67-kDa isoform of glutamic acid decarboxylase (GAD), the synthesizing enzyme for GABA (Akbarian et al., 1995, Volk et al., 2000), have all been observed in post-mortem PFC of schizophrenia patients. These alterations in GABAergic neurotransmission have also been substantiated by microarray analyses of gene expression, which reported that transcript coding for proteins involved in GABA neurotransmission (including GAD67) was consistently reduced in the PFC (Mirnics et al., 2000, Hakak et al., 2001).

Antibodies directed against the calcium-binding proteins parvalbumin (PV), calbindin D-28 (CB) and calretinin (CR) have been used to investigate changes in the subpopulations of PFC interneurons in post-mortem schizophrenia. These chemical markers have been used to identify specific morphological and functional subgroups of GABA neurons (Conde et al., 1994, Gabbot and Bacon, 1996, Kawaguchi and Kubota, 1997). In the PFC, PV-expressing neurons include chandelier and wide arbor basket cells (DeFelipe, 1997, Lund and Lewis, 1993) while CB is predominantly expressed by double bouquet cells and CR by bipolar, double bouquet and Cajal–Retzius cells (Lund and Lewis, 1993, Conde et al., 1994). Each subpopulation of interneuron contributes specifically to the response properties of the principal projection neurons, and altered GABAergic neurotransmission would dramatically affect cortical function (Gupta et al., 2000). The lack of parvalbumin has been shown to affect the development and the function of cortical neuronal circuits (Schwaller et al., 2004). In schizophrenia, the amount of PV-IR, but not CB-nor CR-IR, profiles was significantly reduced (Beasley and Reynolds, 1997, Lewis et al., 2001, Reynolds et al., 2001, Beasley et al., 2002, Hashimoto et al., 2003).

Based on the decrease of glutathione (GSH) levels in the medial PFC and cerebrospinal fluid (Do et al., 1995, Do et al., 2000) and the reduction of gene expression of GSH synthesizing enzymes (Tosic et al., 2004), we have suggested that a deficit in GSH could be a critical vulnerability factor towards schizophrenia (Do et al., 2004). As a main cellular non-protein antioxidant and redox regulator (Meister and Anderson, 1983, Schafer and Buettner, 2001, Castagné et al., 1999), GSH plays a major role in protecting nervous tissue against oxidative stress (Halliwell, 1992, Rabinovic and Hastings, 1998) and modulating redox-sensitive sites including N-methyl d-aspartate (NMDA) receptors (Kohr et al., 1994, Choi and Lipton, 2000). Converging evidence point to the involvement of oxidative stress (Mahadik and Mukherjee, 1996, Herken et al., 2001, Marchbanks et al., 2003, Altar et al., 2005, Prabakaran et al., 2004), NMDA receptor hypofunction (Coyle and Tsai, 2004) and neuronal misconnections during development (Andreasen, 2000, Parnas et al., 1996) as factors contributing to the pathophysiology of schizophrenia (see review in Harrison and Weinberger, 2005). Combination of lowered capacity to regulate oxidative stress by reduced GSH expression and increased generation of reactive oxidant species by elevated extracellular dopamine in regions with dense dopaminergic innervation could lead to focal cellular and regional lesions similar to those reported for brains of schizophrenic patients. To test experimentally this hypothesis, we set up an animal model using the mutant Osteogenic Disorder-Shionogi (ODS) rats, treated with a GSH synthesis inhibitor (Meister, 1995) l-buthionine-(S,R)-sulfoximine (BSO) and with a dopamine reuptake inhibitor (Matecka et al., 1996) 1-(2-(bis-(4-fluorophenyl)mthoxy)-ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (GBR 12909). Treatments were induced during the period of maximal brain growth rate (from PND 5 to 16), when GSH levels were observed to be highest in the rat brain (Nanda et al., 1996). ODS strain was chosen because of their inability to synthesize ascorbic acid (AA), an antioxidant that may compensate for GSH deficit (Martensson and Meister, 1992). In the present study, we focus on a morphological analysis of the three subpopulations of local GABAergic inhibitory interneurons in the cerebral cortex expressing PV, CB or CR.

Section snippets

Animals and treatments

Sixteen Osteogenic Disorder-Shigonagi (ODS) male pups were used in this study. Pregnant female ODS rats were obtained at embryonic days 10–15 from a commercial animal breeder (RCC Limited, Füllinsdorf, Switzerland). Females and their litters were caged under a controlled/light–dark regimen 12/12 h (lights on at 7h00) in plastic Macrolon cages type 4 (595 × 380 × 200 mm, Indulab AG, Gams, Switzerland). Procedures and animal care were in accordance with NIH Guide for the Care and Use of

Results

To determine the effect of GSH deficit and increased extracellular DA during early brain development, immunohistochemistry was performed focusing on markers for GABAergic interneurons (PV, CB and CR). The changed intensity of staining and morphology of immunoreactive neurons was analyzed in series of coronal sections of the whole hemispheres. Once a change in immunolabeling for one of the interneuron markers was identified in a given area, sections of that area where processed for quantitative

Discussion

The main result of the present study is that the number of small parvalbumin-immunoreactive (PV-IR) profiles was markedly reduced in the anterior cingulate (ACG) cortex of rats with glutathione deficit and excess of dopamine during early brain development. Thus, there is a cellular specificity as the parvalbumin (PV) neurons are altered, but not the calbindin (CB) nor calretinin (CR) ones, and area specificity as the change is restricted to ACG. This finding is analogous to the observations

Acknowledgments

The authors wish to thank Dr. Vincent Castagné for his help with animal treatment and “animal keeping”. We are also grateful to Dr. Cécile Lebrand for her helpful advice and David Rodriguez for his help with immunohistochemistry processing. We also thank Prof. Pierre Magistretti for his constant support. This work has been supported by the grants from the Swiss National Foundation 31-62113.00 (to JPH) and 31-55924.98 (to KQD).

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