Fig. 5.

The importance of the C domain in inhibitory synapse formation. (A) Triple-immunofluorescence images of anti-GABA_ARγ2 (red), anti-VGAT (magenta) and anti-EGFP (green) antibodies in cultured hippocampal neurons transfected with EGFP-tagged gephyrin WT and G-E variant. Yellow arrows mark colocalization of gephyrin with both GABA_ARγ2 and VGAT puncta. (Scale bar, 10 μm.) (B) Quantification of inhibitory synaptic targeting of gephyrin variants in cultured hippocampal neurons (2–3 dendrites per transfected neuron were analyzed and group-averaged). (WT and G-E ^****P < 0.0001). Error bars ± SEM. (C) Triple-immunofluorescence images of anti-GABA_ARγ2 (red), anti-VGAT (magenta) and anti-EGFP (green) antibodies in cultured hippocampal neurons transfected with EGFP alone or co-transfected with EGFP and EGFP-tagged gephyrin variants. (Scale bar, 10 μm.) (D) Effects of overexpression of gephyrin WT or the indicated variants on GABA_ARγ2⁺VGAT⁺ puncta density (2–3 dendrites per transfected neuron were analyzed and group averaged). (Ctrl and WT ^**P = 0.0017, WT and G-E ^####P < 0.0001). Error bars ± SEM. (E–G) Representative traces and quantification of frequency and amplitude of mIPSCs recorded from hippocampal cultured neurons transfected with the indicated gephyrin variants. (F) Ctrl and G-E ^*P = 0.0258, WT and G-E ^###P = 0.0004; (G) WT and G-E ^#P = 0.0351). Error bars ± SEM; Control, n = 19; WT, n = 21; G-E, n = 19. (H) Triple-immunofluorescence images of anti-GABA_ARγ2 (red), anti-VGAT (magenta) and anti-EGFP (green) antibodies in cultured hippocampal neurons transfected with EGFP-tagged gephyrin WT and ∆C4 variant. Yellow arrows mark colocalization of gephyrin with both GABA_ARγ2 and VGAT puncta. (Scale bar, 10 μm.) (I) Inhibitory synaptic targeting of gephyrin variants in cultured hippocampal neurons (2–3 dendrites per transfected neuron were analyzed and group-averaged). (WT and ∆C4 ^****P < 0.0001). Error bars ± SEM. (J) Triple-immunofluorescence images of anti-GABA_ARγ2 (red), anti-VGAT (magenta) and anti-EGFP (green) antibodies in cultured hippocampal neurons transfected with EGFP alone or co-transfected with EGFP and EGFP-tagged gephyrin variants. (Scale bar, 10 μm.) (K) Effects of overexpression of gephyrin WT or the indicated variants on GABA_ARγ2⁺VGAT⁺ puncta density (2–3 dendrites per transfected neuron were analyzed and group averaged). (Ctrl and WT ^*P = 0.0365, WT and ΔC4 ^####P < 0.0001). Error bars ± SEM. (L–N) Representative traces and quantification of frequency and amplitude of mIPSCs recorded from hippocampal cultured neurons transfected with the indicated gephyrin variants. [(M) Ctrl and ΔC4 ^**P = 0.0031, WT and ΔC4 ^##P = 0.0015]. Error bars ± SEM; Control, n = 23; WT, n = 28; ΔC4, n = 22.