Elevated GRIA1 mRNA expression in Layer II/III and V pyramidal cells of the DLPFC in schizophrenia

Abstract

The functional integrity of the dorsolateral prefrontal cortex (DLPFC) is altered in schizophrenia leading to profound deficits in working memory and cognition. Growing evidence indicates that dysregulation of glutamate signaling may be a significant contributor to the pathophysiology mediating these effects; however, the contribution of NMDA and AMPA receptors in the mediation of this deficit remains unclear. The equivocality of data regarding ionotropic glutamate receptor alterations of subunit expression in the DLPFC of schizophrenics is likely reflective of subtle alterations in the cellular and molecular composition of specific neuronal populations within the region. Given previous evidence of Layer II/III and V pyramidal cell alterations in schizophrenia and the significant influence of subunit composition on NMDA and AMPA receptor function, laser capture microdissection combined with quantitative PCR was used to examine the expression of AMPA (GRIA1-4) and NMDA (GRIN1, 2A and 2B) subunit mRNA levels in Layer II/III and Layer V pyramidal cells in the DLPFC. Comparisons were made between individuals diagnosed with schizophrenia, bipolar disorder, major depressive disorder and controls (n = 15/group). All subunits were expressed at detectable levels in both cell populations for all diseases as well as for the control group. Interestingly, GRIA1 mRNA was significantly increased in both cell types in the schizophrenia group compare to controls, while similar trends were observed in major depressive disorder (Layers II/III and V) and bipolar disorder (Layer V). These data suggest that increased GRIA1 subunit expression may contribute to schizophrenia pathology.

Introduction

Converging evidence from human postmortem, neuropsychological and imaging studies implicate the dorsolateral prefrontal cortex (DLPFC) as an important brain region in schizophrenia (Bunney and Bunney, 2000, Callicott et al., 2000, Castner et al., 2004, Goldman-Rakic, 1994, Goldman-Rakic and Selemon, 1997, Lewis, 1995, Weinberger et al., 1994, Weinberger et al., 1986). Cognitive dysfunction is a defining feature of schizophrenia (Goldberg et al., 1993, Harvey et al., 2001, Weinberger and Gallhofer, 1997) and is associated with deficits in DLPFC functioning (Goldman-Rakic, 1994, Goldman-Rakic and Selemon, 1997, Lewis and Gonzalez-Burgos, 2000, Weinberger et al., 2001).

The subtlety of structural changes in the DLPFC of individuals diagnosed with schizophrenia (Goldstein et al., 1999, Schlaepfer et al., 1994, Sullivan et al., 1998) suggests that dysfunction in this region may result from alterations in circuitry and cellular connectivity versus gross structural abnormalities. Within DLPFC, Layer III pyramidal cells exhibit decreased cell size (Pierri et al., 2001, Rajkowska et al., 1998), spine density (Glantz and Lewis, 2000, Hill et al., 2006), dendritic arborization and complexity (Kalus et al., 2000), synaptic connectivity (Mirnics et al., 2001, Selemon and Goldman-Rakic, 1999), and increased neuronal density (Selemon et al., 1995, Selemon et al., 1998). Similarly, Layer V pyramidal cells exhibit decreased somal size (Cotter et al., 2002) and dendritic spine density (Black et al., 2004). The involvement of DLPFC Layer II/III and V pyramidal neurons in intrinsic and extrinsic circuitry and the aforementioned anatomical and morphological disruptions of these cells make these cells prime candidates for probing disease-related differences in gene expression associated with schizophrenia.

Substantial evidence indicates a hypofunction of NMDA (see for review (Coyle, 2006)) and AMPA receptors in the brains of individuals diagnosed with schizophrenia (Goff and Coyle, 2001, Goff et al., 2001, Lynch, 2004, O'Neill et al., 2004). NMDA and AMPA receptors are highly abundant in pyramidal cells in the DLPFC (Beneyto and Meador-Woodruff, 2004., Conti et al., 1999, Conti et al., 1994, Huntley et al., 1997, Vickers et al., 1995); however, little is known about the alterations of subunit composition of ionotropic glutamate receptors within this region in individuals diagnosed with schizophrenia. The subunit stoichiometry of NMDA and AMPA receptors determines several of the kinetic and pharmacological properties of the receptor (Cull-Candy et al., 2001, Dingledine et al., 1999). Determining alterations in subunit composition in the schizophrenic brain may provide insight into dysfunctional glutamate signaling in the disease.

Previous studies have examined the expression of NMDA and AMPA receptor subunits in the DLPFC and report increased NR1 (Dracheva et al., 2001), NR2D (Akbarian et al., 1996), and GluR1, decreased GluR2 (Beneyto and Meador-Woodruff, 2006, Vawter et al., 2002), and increased (Dracheva et al., 2005) and/or decreased (Beneyto and Meador-Woodruff, 2006) GluR4 subunit mRNA expression in the DLPFC of schizophrenics. Other studies report no change in NMDA receptor mRNA or protein (Kristiansen et al., 2006) or AMPA receptor subunit mRNA (Healy et al., 1998, O'Connor et al., 2007).

While regional assessments of gene expression create an informative mosaic of expression level changes, the molecular pathology of schizophrenia is likely attributable to dysfunction of discrete components within neuronal circuits in affected brain regions. However, reliance on regional assessment emphasizes gene expression contained in the majority of cells of the neuronal population and/or those genes in highest abundance in the region, which may not adequately reflect alterations in gene expression in target neurons. Microdissection of discrete cell populations, for example by laser capture microdissection (LCM), allows the quantification of multiple transcripts within specific neuronal populations when combined with quantitative gene expression strategies (Backes and Hemby, 2003, Fasulo and Hemby, 2003, Ginsberg et al., 2000, Hemby, 2004, Hemby et al., 2002, Hemby et al., 2003, Kamme and Erlander, 2003, Kamme et al., 2003).

To this end, the combination of LCM and quantitative PCR (qPCR) was used to compare the relative expression levels of NMDA and AMPA subunits in Layers II/III and V pyramidal neurons of the DLPFC in individuals diagnosed with schizophrenia (SCZ), bipolar disorder (BPD), major depressive disorder (MDD) and nonpsychiatric control (CRTL) to determine the specificity of transcriptional changes in NMDA and/or AMPA receptor subunits across these spectrum disorders.