Distribution of alpha1, alpha4, gamma2, and delta subunits of GABAA receptors in hippocampal granule cells

Abstract

GABAA receptors are pentamers composed of subunits derived from the alpha, beta, gamma, delta, theta, epsilon, and pi gene families. alpha1, alpha4, gamma2, and delta subunits are expressed in the dentate gyrus of the hippocampus, but their subcellular distribution has not been described. Hippocampal sections were double-labeled for the alpha1, alpha4, gamma2, and delta subunits and GAD65 or gephyrin, and their subcellular distribution on dentate granule cells was studied by means of confocal laser scanning microscopy (CLSM). The synaptic versus extrasynaptic localization of these subunits was inferred by quantitative analysis of the frequency of colocalization of various subunits with synaptic markers in high-resolution images. GAD65 immunoreactive clusters colocalized with 26.24+/-0.86% of the alpha1 subunit immunoreactive clusters and 32.35+/-1.49% of the gamma2 subunit clusters. In contrast, only 1.58+/-0.13% of the alpha4 subunit immunoreactive clusters and 1.92+/-0.15% of the delta subunit clusters colocalized with the presynaptic marker GAD65. These findings were confirmed by studying colocalization with immunoreactivity of a postsynaptic marker, gephyrin, which colocalized with 27.61+/-0.16% of the alpha1 subunit immunoreactive clusters and 23.45+/-0.32% of the gamma2 subunit immunoreactive clusters. In contrast, only 1.90+/-0.13% of the alpha4 subunit immunoreactive clusters and 1.76+/-0.10% of the delta subunit clusters colocalized with gephyrin. These studies demonstrate that a subset of alpha1 and gamma2 subunit clusters colocalize with synaptic markers in hippocampal dentate granule cells. Furthermore, all four subunits, alpha1, alpha4, gamma2, and delta, are present in the extrasynaptic locations.

Fig. 1

Characterization of GABA_A receptor subunit α1, γ2, α4, and δ by Western blot. (A) A single 51 kDa band of α1. (B) A 67 kDa band of α4. (C) A 42 kDa band of γ2 was seen. (D) A 54 kDa δ band was shown.

Fig. 2

Distribution of GABA_A receptor α1, γ2, α4, and δ subunit immunoreactivity in hippocampal dentate granule cells acquired by CLSM, 40× objective lens, scale bar =30 μm (shown in A). Sections were labeled with GABA_A subunit isoform-specific antibodies and immunoreactivity was assessed in the form of clusters that outlined dentate granule cell soma. The immunoreactivity of α1 (A) and γ2 (B) subunits was similar, the somata and dendrites of interneurons near the hilar border were strongly labeled (A, B; arrowhead); fewer clusters were found in granule cell somata, molecular, and granule cell layers. The immunoreactivity of α4 (C) and δ (D) subunits was more diffuse and clusters were less obvious. Some interneurons in the molecular layer stained intensely for the δ subunit. Intense staining of the δ subunit was found in the molecular layer.

Fig. 3

Distribution of GAD65 (A) and gephyrin (B) immunoreactivity in the dentate gyrus, scale bar =30 μm. There was intense punctate GAD65 staining in the granule cell layer and in the inner molecular layer, which formed a net of clusters (A). In the somata of dentate granule cells, there was diffuse low intensity gephyrin immunoreactivity and more intense punctate staining (B).

Fig. 4

Large clusters of α1 or γ2 GABA_A receptor subunits frequently colocalized with synaptic makers (arrowhead), whereas most small clusters did not (arrow). Hippocampal dentate granule cells were double-labeled with the presynaptic marker GAD (B, E) or the postsynaptic marker gephyrin (H, K) and GABA_A subunit isoform-specific antibodies to α1 (A, G) or γ2 (D, J). Merged images of the markers and subunits are shown in C, F, I, and L. GABA_A subunits were visualized by secondary antibody conjugated with Alexa Fluor 488 (green) and synaptic markers with Alexa Fluor 594 (red). Scale bar =5 μm.

Fig. 5

Percent colocalizaton of different GABA_A receptor subunits with the synaptic markers GAD65 and gephyrin is demonstrated. The percent fraction colocalization of GABA_A receptor α1 or γ2 subunits with GAD65 was significantly higher than those of α4 or δ subunits, respectively (*p<0.0001, A). The percent fraction of colocalization of GABA_A receptor α1 or γ2 with gephyrin are significantly higher than those of α4 or δ subunits, respectively (*p<0.0001, B).

Fig. 6

Clusters of α4 and δ GABA_A receptor subunits rarely colocalized with synaptic makers. Hippocampal dentate granule cells were double-labeled with the presynaptic marker GAD (B, E) or the postsynaptic marker gephyrin (H, K) and GABA_A subunit isoform-specific antibodies to α4 (A, G) or δ (D, J). GABA_A subunits were visualized by secondary antibody conjugated with Alexa Fluor 488 (green) and synaptic markers with Alexa Fluor 594 (red). Merged images of synaptic markers and subunits are shown in C, F, I, and L. Scale bar =5 μm.