Expression of mouse P304L and human P311L mutant HGSNAT variants aggravates GABAergic synaptic defects in cultured primary hippocampal mouse neurons. (A) Hippocampal neurons from Hgsnat^P304L and Hgsnat-Geo mice show reduction in density of Syn1⁺ puncta in proximity to MAP2⁺ dendrites compared with WT cells. The levels of Syn1⁺ puncta are rescued by expression of WT active human HGSNAT but not of the P311L variant. (B) Hippocampal neurons from Hgsnat^P304L and Hgsnat-Geo mice show an equal reduction in density of PSD-95⁺/VGLUT1⁺ puncta in juxtaposition. The levels of PSD-95⁺/VGLUT1⁺ puncta in juxtaposition are rescued by expression of WT active human HGSNAT but not of the P311L variant. (C) Densities of Gephyrin⁺/VGAT⁺ puncta in juxtaposition are reduced in neurons from Hgsnat^P304L mice but not from Hgsnat-Geo mice compared with WT cells. Primary hippocampal neurons of Hgsnat-Geo mice transduced with LV-P311L-HGSNAT show reduction of Gephyrin⁺/VGAT⁺ puncta in juxtaposition compared with WT and nontransduced Hgsnat-Geo cells. The panels show representative confocal images of neurons and enlargements of selected axonal and dendritic fragments. Scale bars equal 40 μm. Graphs show results of puncta quantification with ImageJ software. Puncta were quantified in 20-μm-long segments of dendrite or axon, 30 μm away from the neuronal soma. Individual data, means, and SD from three independent cultures, each involving pooled embryos from at least three mice per genotype, are shown. For each culture, 5–10 neurons were analyzed. P values were calculated using nested one-way ANOVA test with Tukey post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.