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. 2019 Jun 4;13:217. doi: 10.3389/fncel.2019.00217

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Copyright © 2019 Duan, Pandey, Li, Castellano, Gu, Li, Tian and Lu.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Loss of GABA_ARs strongly reduced inhibitory synapse density in hippocampal neurons. (A–C) Single-cell KO of GABA_ARs significantly reduced gephyrin (red) and vGAT (magenta) puncta as well as co-localization of gephyrin and vGAT in hippocampal neurons. (A) Representative images of gephyrin and vGAT-immunolabeling in neurons expressing GFP (top) or β1-3 gRNAs (bottom). (B) Bar graphs showed the quantitation of gephyrin (top), vGAT (middle), and co-localization of gephyrin and vGAT (bottom) in neuronal somata (gephyrin density and size: GFP, n = 22, β1-3 gRNAs, n = 24; ^∗∗∗∗p < 0.0001; vGAT density and size: GFP, n = 22, β1-3 gRNAs, n = 24, ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001; co-localization: percentage of gephyrin puncta colocalized with vGAT (gephyrin⁺ vGAT) (GFP, n = 22, β1-3 gRNAs, n = 24; ^∗∗p < 0.01) and percentage of vGAT puncta colocalized with gephyrin (vGAT⁺ gephyrin) (GFP, n = 22, β1-3 gRNAs, n = 24; ^∗∗p < 0.01); t-test; N = 5). (C) Bar graphs showed the quantitation of gephyrin (top), vGAT (middle), and co-localization of gephyrin and vGAT (bottom) in neuronal dendrites (gephyrin density and size: GFP, n = 63, β1-3 gRNAs, n = 66, ^∗∗∗∗p < 0.0001; vGAT density and size: GFP, n = 22, β1-3 gRNAs, n = 24, ^∗∗∗∗p < 0.0001, ^∗∗p < 0.01; co-localization: percentage of gephyrin puncta colocalized with vGAT (gephyrin⁺ vGAT) (GFP, n = 35, β1-3 gRNAs, n = 36; ^∗∗p < 0.01) and percentage of vGAT puncta colocalized with gephyrin (vGAT⁺ gephyrin) (GFP, n = 35, β1-3 gRNAs, n = 36; ^∗p < 0.05); t-test; N = 6). GFP was not immunolabeled with anti-GFP antibodies. Scale bar, 10 μm for whole cell, 5 μm for somata and dendrite. (D) Neuroligin2 (NL2) puncta were not changed in hippocampal neurons expressing β1-3 gRNAs (GFP, n = 19, β1-3 gRNAs, n = 21, p > 0.05, t-test; N = 3). GFP was immunolabeled with anti-GFP antibodies to boost the fluorescence (green). Scale bar, 5 μm. n represents the number of cells analyzed and N represents the number of independent experiments.