AMPA receptor subunit and splice variant expression in the DLPFC of schizophrenic subjects and rhesus monkeys chronically administered antipsychotic drugs

Abstract

Disturbances in glutamate neurotransmission are thought to be one of the major contributing factors to the pathophysiology of schizophrenia. In the dorsolateral prefrontal cortex (DLPFC), glutamate neurotransmission is largely mediated by AMPA receptors. Data regarding alterations of subunit expression in the brains of patients with schizophrenia remain equivocal. This may be due to differences in technique sensitivity, endogenous control selection for normalization of data, or effect of antipsychotic drug treatment in different cohorts of schizophrenia. This study attempted to address these issues by examining the expression of AMPA receptor subunits and splice variants in the DLPFC of two schizophrenia cohorts using quantitative PCR (qPCR) with normalization to the geometric mean of multiple endogenous controls. In addition, a non-human primate model of chronic antipsychotic drug administration was used to determine the extent to which the transcript expression may be altered by antipsychotic drug treatment in the primate DLPFC. AMPA receptor subunits and flip and/or flop splice variants were not significantly different in the DLPFC of schizophrenia subjects versus controls in either of the two cohorts. However, in rhesus monkeys chronically treated with antipsychotic drugs, clozapine treatment significantly decreased GRIA1 and increased GRIA3 mRNA expression, while both clozapine and haloperidol increased the expression of GRIA2 subunit mRNA. Expression of AMPA receptor splice variants was not significantly altered by antipsychotic drug administration. This is the first study to show that AMPA receptor subunit mRNAs in the primate DLPFC are altered by antipsychotic drug administration. Antipsychotic drug induced alterations may help explain differences in human post-mortem studies regarding AMPA receptor subunit expression and provide some insight into the mechanism of action of antipsychotic drugs.

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

“…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).…”

“…Sections selected for the LCM were adjacent to those used for regional analysis published previously (O'Connor et al, 2007). Slides were sub-merged in 95% ethanol (2×), and 100% EtOH for 30 s each, followed by submersion in xylene for 5 min and air dried.…”

“…Resulting RNA was quantified using the Quant-iT Ribo Green RNA Assay Kit (Invitrogen #R11490). RNA quality was assessed for each subject from sections adjacent to those used in this study using the Agilent 2100 Bioanalyzer with RNA 6000 Nano Lab Chips (O'Connor et al, 2007). Reverse transcription separately on total RNA (75 ng) for Layer II/III and Layer V captured pyramidal cells for each subject using random hexamers and SuperScript III (Invitrogen, Carlsbad, CA).…”

“…Methods for qPCR have been described previously (O'Connor et al, 2007). Standard Taqman assays were purchased for measurement of AMPA and NMDA subunit levels and endogenous controls (Applied Biosystems; GRIA1, Hs00181348_m1; GRIA2, Hs00181331_m1; GRIA3, Hs00241485_m1; GRIA4, Hs00168163_m1; phosphoglycerate kinase 1: PGK1, Hs99999906_m1; peptidylprolyl isomerase A (cyclophilin A): PPIA, Hs99999904_m1; glyceraldehyde-3-phosphate dehydrogenase: GAPDH, Hs99999905_m1; TATA box binding protein, Hs99999910_m1; β-2 microglobulin; Hs99999907_m1; ribosomal protein, large, PO; Hs99999902_m1; ribosomal protein 18 S, Hs99999901_m1; β-actin, Hs99999903_m1; hypoxanthine phosphoribosyltransferase 1, Hs99999909_m1; transferrin receptor, Hs99999911_m1; β-glucuronidase, Hs99999908_m1).…”

“…Data for each gene of interest were expressed as Qty mean/ geometric mean of Qty mean values for the selected endogenous control genes as described previously (O'Connor et al, 2007). Normalized values were expressed as a percent control.…”