Increases in [3H]muscimol and [3H]flumazenil binding in the dorsolateral prefrontal cortex in schizophrenia are linked to α4 and γ2S mRNA levels respectively

Abstract

Background: GABA(A) receptors (GABA(A)R) are composed of several subunits that determine sensitivity to drugs, synaptic localisation and function. Recent studies suggest that agonists targeting selective GABA(A)R subunits may have therapeutic value against the cognitive impairments observed in schizophrenia. In this study, we determined whether GABA(A)R binding deficits exist in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia and tested if changes in GABA(A)R binding are related to the changes in subunit mRNAs. The GABA orthosteric and the benzodiazepine allosteric binding sites were assessed autoradiographically using [(3)H]Muscimol and [(3)H]Flumazenil, respectively, in a large cohort of individuals with schizophrenia (n = 37) and their matched controls (n = 37). We measured, using qPCR, mRNA of β (β1, β2, β3), γ (γ1, γ2, γ2S for short and γ2L for long isoform, γ3) and δ subunits and used our previous measurements of GABA(A)R α subunit mRNAs in order to relate mRNAs and binding through correlation and regression analysis.

Results: Significant increases in both [(3)H]Muscimol (p = 0.016) and [(3)H]Flumazenil (p = 0.012) binding were found in the DLPFC of schizophrenia patients. Expression levels of mRNA subunits measured did not show any significant difference in schizophrenia compared to controls. Regression analysis revealed that in schizophrenia, the [(3)H]Muscimol binding variance was most related to α4 mRNA levels and the [(3)H]Flumazenil binding variance was most related to γ2S subunit mRNA levels. [(3)H]Muscimol and [(3)H]Flumazenil binding were not affected by the lifetime anti-psychotics dose (chlorpromazine equivalent).

Conclusions: We report parallel increases in orthosteric and allosteric GABA(A)R binding sites in the DLPFC in schizophrenia that may be related to a "shift" in subunit composition towards α4 and γ2S respectively, which may compromise normal GABAergic modulation and function. Our results may have implications for the development of treatment strategies that target specific GABA(A)R receptor subunits.

Figure 1. An overview of the experimental procedures used in the autoradiography experiments.

A Sagittal view of brain with lines indicating area of coronal section with the blue outlined area is where homogenate tissue was isolated with the dashed lines indicating the bounds of tissue slices used for autoradiography (A). Typical autoradiographs (B) obtained with [³H]Muscimol (GABA orthosteric binding site agonist) and [³H]Flumazenil (benzodiazepine allosteric binding site antagonist) in the DLPFC of control (CON) and schizophrenia (SCZ) cases. NeuN staining (C) that allowed identification of the cortical layers in the DLPFC is presented on the right side of the figure, along with the drawing methodology used to quantify binding in the “superficial” (layers I, II, and III) and “deep” layers (layers IV, V and VI) of the DLPFC. The superficial layers quantification “box” is indicated in solid line, while the deep layers quantification “box” is indicated in dotted line.

Figure 2. Scatter plots presenting the binding values (fmoles/mg tissue equivalent) of [³H]Muscimol (A) and [³H]Flumazenil (B) in control (CON) (n = 37) and schizophrenia (SCZ) (n = 37), in the superficial (surface) and deep layers of the DLPFC.

* = p<0.05 and refers to a significant main effect of diagnosis (schizophrenia>control); # = p<0.001 and refers to a significant main effect of layers (superficial>deep) after two-way ANCOVA. Bars represent the means.

Figure 3. Scatter plots of the normalised expression level of GABA_AR mRNA subunits (qRT-PCR) in the DLPFC of control (CON) (n = 37) and schizophrenia (SCZ) (n = 37) cases.

(A) β mRNA subunits and (B) γ and δ mRNA subunits. Bars represent the means.

Figure 4. Correlations of [³H]Muscimol binding and α4 mRNA subunit expression level in the superficial layers of the DLPFC of control (CON) (n = 37) and schizophrenia (SCZ) (n = 37) cases.

Figure 5. Correlations of [³H]Flumazenil binding and γ2S mRNA subunit expression level in the superficial layers of the DLPFC of control (CON) (n = 37) and schizophrenia (SCZ) (n = 37) cases.