Fig. 4

ARF6 activity is required for GABAergic synapse development in vivo. a Schematic diagram of AAV vectors expressing sh-Arf6 and HA-tagged ARF6 and its mutants (T27 N and T157A) used in c–g. b Immunoblotting analyses with ARF6 antibodies showing the KD efficacy of sh-ARF6 in vivo. Lysates from mouse brain stereotactically injected with AAVs expressing sh-ARF6 were collected and immunoblotted with anti-ARF6 antibodies. Anti-β-actin antibodies were used as normalization controls. c Representative images illustrating EGFP expression after AAV injection into the hippocampal DG region. Brain sections were immunostained for EGFP (green) or HA (red) and counterstained with DAPI (blue). Scale bar: 20 μm (applies to all images). d Representative images showing GABA_ARγ2⁺ puncta in the DG of mice stereotactically injected with AAVs expressing Control or sh-Arf6, or coexpressing sh-Arf6 and the indicated ARF6 variants (ARF6-WT, ARF6-T27 N, or ARF6-T157A). Scale bar, 20 μm (applies to all images). Abbreviations: MOL, molecular layer; GCL, granule cell layer. e Quantification of the density and size of GABA_ARγ2⁺ puncta per tissue area. Data are presented as means ± SEMs (n = 20–25 sections/4–5 mice; *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; non-parametric ANOVA with Kruskal-Wallis test followed by post hoc Dunn’s multiple comparison test). f Representative images of AAV-infected neurons in DG molecular and hilar regions immunostained for the excitatory marker VGLUT1. Scale bar: 20 μm (applies to all images). g Quantification of VGLUT1⁺ puncta intensity per tissue area. Data are presented as means ± SEMs from 3 to 5 independent experiments (n = 22–30 sections/4–6 mice; *p < 0.05, **p < 0.01 vs. control; non-parametric ANOVA with Kruskal-Wallis test, followed by post hoc Dunn’s multiple comparison test)