Activity-Dependent Inhibitory Synapse Scaling Is Determined by Gephyrin Phosphorylation and Subsequent Regulation of GABAA Receptor Diffusion

Sereina Battaglia; Marianne Renner; Marion Russeau; Etienne Côme; Shiva K. Tyagarajan; Sabine Lévi

doi:10.1523/ENEURO.0203-17.2017

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Figure 1.
Morphologic characterization of eGFP-gephyrin and its mutant variants. A, Representative images of primary hippocampal neurons cotransfected with eGFP-WT, eGFP-S268E, eGFP-S270A, or eGFP-DN and shRNA-3′UTR. eGFP-gephyrin clusters (green), α2 GABA_ARs (red), and VGAT (blue) are shown. Scale bar, 10 μm. B, Quantification of eGFP-gephyrin cluster density, cluster area, and intensity shows larger eGFP-S270A clusters compared with eGFP-WT at synapses. S268E: WT n = 66 cells, S268E n = 60 cells, 4 cultures. Syn: Cluster Number (Nb) p = 0.42, area p = 0.22, intensity p = 0.05. Extra: Nb p = 0.99, Area p = 0.66, Intensity p = 0.44. S270A: WT n = 86 cells, S270A: n = 74 cells, 6 cultures. Syn: Nb p = 0.77, Area p = 0.02, Intensity p = 0.02. Extra: Nb p = 0.39, Area p = 0.42, Intensity p = 0.15. C, Quantification for α2 GABA_AR clusters shows significantly more receptors in eGFP-S270A mutant clusters. S268E: WT n = 52 cells, S268E n = 47 cells, 3 cultures. Syn: Nb p = 0.48, Area p = 0.46, Intensity p = 0.6. Extra: Nb p = 0.46, area p = 0.63, intensity p = 0.22. S270A: WT n = 52 cells, S270A n = 39 cells, 3 cultures. Syn: Nb p = 0.56, Area p = 0.08, Intensity p = 0.008. Extra: Nb p = 0.008, Area p = 0.81, Intensity p = 0.29. Data shown as mean ± SEM. Values were normalized to the corresponding control values. Statistics *, p ≤ 0.05, **, p ≤ 0.01 (Mann–Whitney rank sum test).
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Figure 2.
Membrane dynamics of α2 GABA_AR is influenced by gephyrin phosphorylation. A, Example traces of QD trajectories (red) overlaid with fluorescent synaptic clusters (white) of VGAT-Oyster550 for eGFP-DN transfected neurons or with eGFP-gephyrin clusters for eGFP-WT, eGFP-S268E, or eGFP-S270A expressing cells. Scale bar, 0.5 µm. B, Median diffusion coefficients D of α2 GABA_AR in neurons transfected with either eGFP-WT or eGFP-DN. Extra: WT n = 975 QDs, DN n = 491 QDs, p = 4.5 × 10⁻³⁴; Syn: WT n = 306 QDs, DN n = 173 QDs, p = 0.36. C, Quantification of explored area EA of α2 GABA_AR, Extra: WT n = 2925 QDs, DN n = 1473 QDs, p = 3.8 × 10⁻²³; Syn: WT n = 918 QDs, DN n = 519 QDs, p = 4.4 × 10⁻⁴. D, Dwell time DT of α2 GABA_AR at synapses in neurons transfected with either eGFP-WT or eGFP-DN. Quantification of all QDs (total), trapped (DT < 5.9 s), and passing (DT > 5.9 s) QDs at inhibitory synapses. Significant decrease in synaptic dwell time for total and trapped QDs was observed but not for passing ones. Total: WT n = 436 QDs, DN n = 262 QDs, p = 0.001; Trapped: WT n = 235 QDs, DN n = 108 QDs, p = 8.0 × 10⁻³; Passing: WT n = 201 QDs, DN n = 154 QDs, p = 0.19. E, Quantification of diffusion coefficients of α2 GABA_AR showing increased receptor mobility at extrasynaptic (extra) and synaptic (syn) sites in neurons transfected with eGFP-S268E or eGFP-S270A, compared with eGFP-WT expressing cells. Extra: WT n = 1820 QDs, S268E n = 1273 QDs, p = 1.1 × 10⁻²², S270A n = 1658, p = 2.9 × 10⁻²⁷. Syn: WT n = 461 QDs, S268E n = 326 QDs, p = 2.4 × 10⁻⁸, S270A n = 340, p = 1.8 × 10⁻⁸. F, Quantification of α2 GABA_AR explored area EA, Extra: WT n = 5460 QDs, S268E n = 3807 QDs, p = 6.8 × 10⁻⁵², S270A n = 5355, p = 2.2 × 10⁻¹⁰¹. Syn: WT n = 1383 QDs, S268E n = 978 QDs, p = 7.4 × 10⁻²³, S270A n = 2208, p = 1.2 × 10⁻³³. G, Quantification of α2 GABA_AR dwell time DT in neurons expressing eGFP-WT, eGFP-S268E, or eGFP-S270A. Calculations were done for all QDs (total), (trapped), or (passing) QDs at inhibitory synapses. Decrease in dwell time for the whole or trapped population of QDs was seen in synapses expressing eGFP-S268E but not in synapses containing eGFP-S270A. Total: WT n = 251 QDs, S268E n = 176 QDs, p = 0.013, S270A n = 216 QDs, p = 0.31; Trapped: WT n = 135 QDs, S268E n = 85 QDs, p = 0.002, S270A n = 109 QDs, p = 0.28; Passing: WT n = 116 QDs, S268E n = 91 QDs, p = 0.24, S270A n = 107 QDs, p = 0.98. All data are from six independent experiments. In B, C, E, and F, data are presented as median values ± 25%–75% IQR, ***, p ≤ 0.001 (Kolmogorov–Smirnov test). In D and G, data are presented as mean ± SEM. *, p ≤ 0.05, **, p ≤ 0.01 (Mann–Whitney rank sum test). D in µm²/s, EA in µm², DT in s.
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Figure 3.
PALM imaging showing gephyrin phosphorylation influences scaffold packing. A, Epifluorescence (top) and PALM (bottom) imaging of the same dendritic regions in neurons expressing pDendra2-WT, -S268E, or -S270A mutant. Scale bar, 1 µm. B, Representative image of cluster segmentation (red) based on local density of molecules detected (white dots) using a threshold of 1000 detections/µm² (blue). Scale bar, 200 nm. C, Quantification of eGFP cluster area using PALM shows reduction in cluster size for eGFP-S268E and increase in cluster size for eGFP-S270A compared with eGFP-WT. WT n = 313 synapses, S268E n = 277 synapses, S270A n = 290 synapses, p < 0.001, 4 cultures. D, Quantification of density of gephyrin molecules per square micrometer using PALM in transfected neurons. Neurons expressing eGFP-S268E exhibit denser gephyrin packing, and neurons expressing eGFP-S270A exhibit less dense packing of gephyrin compared with eGFP-WT. Data are presented as mean ± SEM. **, p = 0.006; ***, p ≤ 0.001 (Mann–Whitney rank sum test).
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Figure 4.
Gephyrin clustering influences GABA_AR lateral diffusion. A, Morphology of eGFP-WT (green) after 8 and 48 h of 4-AP application; VGAT (blue), GABA_AR α2 (red) at 21 DIV. Scale bar, 10 µm. B, Quantification of eGFP-WT clusters after 8 and 48 h of 4-AP application. t0 n = 55 cells, 8 h n = 46 cells, 48 h n = 55 cells, 3 cultures. Cluster Nb: 0–8 h: p = 0.13, 0–48 h: p = 0.002; Area: 0–8 h: p = 0.5, 0–48 h: p = 0.001; Intensity: 0–8 h: p < 0.001, 0–48 h: p < 0.001. C, Quantification of synaptic α2 GABA_AR clusters after 8 and 48 h of 4-AP compared with mock treated control. t0 n = 52 cells, 8 h n = 43 cells, 48 h n = 53 cells, 3 cultures. Cluster Nb: 0–8 h: p = 0.4, 0–48 h: p = 0.3; Area: 0–8 h: p = 0.8, 0–48 h: p = 0.8; Intensity: 0–8 h: p = 0.5, 0–48 h: p = 0.03. D, Quantification of extrasynaptic α2 GABA_AR clusters after 8 and 48 h of 4-AP compared with mock treated control. t0 n = 52 cells, 8 h n = 43 cells, 48 h n = 53 cells, 3 cultures. Cluster Nb: 0–8 h: p = 0.2, 0–48 h: p = 0.9; Area: 0–8 h: p = 0.02, 0–48 h: p = 0.3; Intensity: 0–8 h: p = 0.05, 0–48 h: p = 0.022. E, Example trace of α2 GABA_AR trajectories showing surface exploration of extrasynaptic and synaptic receptors after 8 and 48 h of 4-AP exposure. Scale bar, 0.5 µm. F, Quantification of diffusion coefficients of α2 GABA_AR after 8 h of 4-AP exposure. Extra; t0 n = 450 QDs, WT 4AP 8 h n = 961 QDs, p = 1.96 10⁻⁷. Syn; t0 n = 103 QDs, 8 h n = 138 QDs, p = 0.22; 2 cultures. G, Quantification of explored area EA of α2 GABA_AR after 8 h of 4-AP application. Extra; t0 n = 1347 QDs, 8 h n = 5265 QDs, p = 6.4 × 10⁻⁹. Syn; t0 n = 308 QDs, 8 h n = 708 QDs, p = 0.63. H, Quantification of synaptic dwell time DT of α2 GABA_AR showing no impact after 8 h of 4-AP for total, trapped, or passing receptor population. Total: t0 n = 151 QDs, 8 h n = 206 QDs, p = 0.073; Trapped: t0 n = 80 QDs, 8 h n = 116 QDs, p = 0.36; Passing: t0 n = 78 QDs, 8 h n = 90 QDs, p = 0.02. I, Quantification of diffusion coefficients of α2 GABA_AR after 48 h of 4-AP application. Extra: t0 n = 777 QDs, 48 h n = 174 QDs, p = 0.69. Syn: t0 n = 126 QDs, 48 h n = 213 QDs, p = 1.4 × 10⁻⁴. J, Quantification of explored area EA of α2 GABA_AR after 48 h of 4-AP application. Extra: t0 n = 2331 QDs, 48 h n = 5508 QDs, p = 0.045. Syn: t0 n = 378 QDs, 48 h n = 717 QDs, p = 2.2 × 10⁻²⁰. K, Quantification of α2 GABA_AR dwell time after 48 h of 4-AP application. Total: t0 n = 201 QDs, 48 h n = 254 QDs, p = 0.74. Trapped: t0 n = 91 QDs, 48 h n = 110 QDs, p = 0.99. Passing: t0 n = 110 QDs, 48 h n = 144 QDs, p = 0.81. In B–D, H, and K, data are presented as mean ± SEM, *, p ≤ 0.05; ***, p ≤ 0.001 (Mann–Whitney rank sum test). In F, G, I, and J, data are presented as median values ± 25%–75% IQR, *, p ≤ 0.05; ***, p ≤ 0.001 (Kolmogorov–Smirnov test). In B–G, I, and J, values were normalized to the corresponding control values. In H and K, DT in s.
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Figure 5.
PKA and CaMKIIα signaling pathways regulate gephyrin clustering and α2 GABA_AR membrane dynamics in conditions of chronic changes of activity. A, Morphologic analysis of neurons transfected with eGFP-S303A/S305A (eGFP-SSA) gephyrin double mutant insensitive to PKA and CaMKIIα signaling pathways. Double staining of VGAT (blue) and α2 GABA_AR (red) at 21 DIV under control condition (t0) or in the presence of 4-AP for 48 h. Scale bar, 10 µm. B, Quantifications of synaptic eGFP-SSA clusters and synaptic (α2 syn) and extrasynaptic (α2 extra) α2 GABA_AR clusters in relation to eGFP-WT show minor impact of eGFP-SSA under control condition. eGFP-WT: n = 89 cells, eGFP-SSA n = 95 cells, 6 cultures. eGFP-SSA: Cluster Nb: p = 0.3; Area: p = 0.9; Intensity: p = 0.5. α2 syn: Cluster Nb: p = 0.4; Area: p = 0.5; Intensity: p = 0.8. α2 extra: Cluster Nb: p = 0.2; Area: p = 0.4; Intensity: p = 0.2. C, Quantification of median diffusion coefficient D of α2 GABA_AR in neurons expressing eGFP-WT or eGFP-SSA under control condition. Extra: WT n = 1166 QDs, SSA n = 989 QDs, p = 1.5 × 10⁻¹²; Syn: WT n = 312 QDs, SSA n = 245 QDs, p = 0.08; 4 cultures. D, Quantification of median explored area EA of α2 GABA_AR in neurons expressing eGFP-WT or eGFP-SSA under control condition. Extra: WT n = 3510 QDs, SSA n = 2778 QDs, p = 3.9 × 10⁻¹⁸; Syn: WT n = 932 QDs, SSA n = 735 QDs, p = 3.1 × 10⁻⁴. E, Quantification of α2 GABA_AR dwell time DT at synaptic sites in neurons expressing either eGFP-WT or eGFP-SSA. Calculations were done for all QDs (total), (trapped), or (passing) QDs at inhibitory synapses. No significant differences were found between eGFP-WT and eGFP-SSA. Total: WT n = 390 QDs, SSA n = 335 QDs, p = 0.2; Trapped: WT n = 229 QDs, SSA n = 173 QDs, p = 0.4; Passing: WT n = 161 QDs, SSA n = 162 QDs, p = 0.9. F, Quantification of eGFP-SSA clusters after 8 and 48 h of 4-AP application. t0 n = 61 cells, 8 h n = 52 cells, 48 h n = 93 cells, 3–6 cultures. Cluster Nb: 0–8 h: p = 0.2, 0–48 h: p < 0.001; Area: 0–8 h: p = 0.8, 0–48 h: p = 0.3; intensity: 0–8 h: p = 0.8, 0–48 h: p = 0.2. G, Quantification of synaptic α2 GABA_AR clusters after 8 and 48 h of 4-AP compared with mock treated control. t0 n = 53 cells, 8 h n = 50 cells, 48 h n = 69 cells, 3–6 cultures. Cluster Nb: 0–8 h: p < 0.001, 0–48 h: p < 0.001; Area: 0–8 h: p = 0.002, 0–48 h: p = 0.09; Intensity: 0–8 h: p = 0.5, 0–48 h: p = 0.5. H, Quantification of extrasynaptic α2 GABA_AR clusters after 8 and 48 h of 4-AP compared with mock treated control. Cluster Nb: 0–8 h: p = 0.2, 0–48 h: p = 0.1; Area: 0–8 h: p = 0.01, 0–48 h: p = 0.9; Intensity: 0–8 h: p = 0.002, 0–48 h: p < 0.001. I, Quantification of α2 GABA_AR diffusion coefficients in eGFP-SSA expressing cells after 8 h of 4-AP exposure. Extra: t0 n = 787 QDs, 4AP 8 h n = 365 QDs, p = 3.6 × 10⁻⁴. Syn: t0 n = 212 QDs, 8 h n = 187 QDs, p = 0.4; 5 cultures. J, Quantification of explored area EA of α2 GABA_AR after 8 h of 4-AP application. Extra: t0 n = 1869 QDs, 8 h n = 1092 QDs, p = 0.002. Syn: t0 n = 753 QDs, 8 h n = 558 QDs, p = 0.09. K, Quantification of α2 GABA_AR diffusion coefficients in eGFP-SSA expressing cells after 48 h of 4-AP exposure. Extra: t0 n = 1098 QDs, 4AP 48 h n = 734 QDs, p = 0.002. Syn: t0 n = 287 QDs, 48 h n = 198 QDs, p = 0.2; 5 cultures. L, Quantification of explored area EA of α2 GABA_AR after 48 h of 4-AP application. Extra: t0 n = 2169 QDs, 48 h n = 1500 QDs, p = 0.04. Syn; t0 n = 633 QDs, 48 h n = 510 QDs, p = 0.002. M, Quantification of α2 GABA_AR dwell time DT in neurons expressing eGFP-SSA after 8 or 48 h of 4-AP application. Calculations were done for trapped or passing QDs at inhibitory synapses. Trapped: 8 h: n = 189 QDs, p = 0.3; 48 h: n = 166 QDs, p = 0.1; Passing: 8 h: n = 76 QDs, p = 0.3; 48 h: n = 132 QDs, p = 0.9. In B, E, F–H, and M, data are presented as mean ± SEM. **, p < 0.01; ***, p ≤ 0.001 (Mann–Whitney rank sum test). In C, D, I, and L, data are presented as median values ± 25%–75% IQR; ***, p ≤ 0.001 (Kolmogorov–Smirnov test). In all graphs except E, values were normalized to the corresponding control values.
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Figure 6.
The ERK1/2 pathway does not influence structural synaptic adaptation. A, Morphologic analysis of eGFP-S268E in control (t0) or after 4-AP application for 8 or 48 h. Scale bar, 10 µm. B, Quantification of eGFP-S268E clusters after 8 or 48 h of 4-AP application. t0 n = 50 cells, 8 h n = 54 cells, 48 h n = 55 cells, 3 cultures. Cluster Nb: 0–8 h: p = 0.2, 0–48 h: p = 0.004; Area: 0–8 h: p = 0.02, 0–48 h: p < 0.001; Intensity: 0–8 h: p = 0.003, 0–48 h: p < 0.001. 3 cultures. C, Quantification of synaptic α2 GABA_AR clusters after 8 h and 48 h of 4-AP compared with mock treated control. t0 n = 47 cells, 8 h n = 50 cells, 48 h n = 62 cells, 3-4 cultures. Cluster Nb: 0–8 h: p = 0.08, 0–48 h: p = 0.5; Area: 0–8 h: p = 0.8, 0–48 h: p = 0.03; Intensity: 0–8 h: p = 0.5, 0–48 h: p < 0.001. D, Quantification of extrasynaptic α2 GABA_AR clusters after 8 and 48 h of 4-AP compared with mock treated control. Cluster Nb: 0–8 h: p = 0.006, 0–48 h: p = 0.007; Area: 0–8 h: p = 0.02, 0–48 h: p < 0.001; Intensity: 0–8 h: p = 0.04, 0–48 h: p < 0.001. E, Example traces of α2 GABA_AR trajectories at extrasynaptic (extra) and synaptic (syn) sites under control condition (t0) or after 8 or 48 h of 4-AP application. Scale bar, 0.25 µm. F, Quantification of α2 GABA_AR diffusion coefficients after 8 h of 4-AP exposure. Extra: t0 n = 1230 QDs, 4AP 8 h n = 1855 QDs, p = 3.4 × 10⁻⁶. Syn: t0 n = 281 QDs, 8 h n = 378 QDs, p = 0.2; 3 cultures. G, Quantification of explored area EA of α2 GABA_AR after 8 h of 4-AP application. Extra: t0 n = 3402 QDs, 8 h n = 2454 QDs, p = 3.2 × 10⁻²³. Syn: t0 n = 843 QDs, 8 h n = 984 QDs, p = 0.02. H, Quantification of α2 GABA_AR diffusion coefficients after 48 h of 4-AP exposure. Extra: t0 n = 687 QDs, 4AP 48 h n = 1611 QDs, p = 0.4. Syn: t0 n = 73 QDs, 48 h n = 46 QDs, p = 1.6 × 10⁻⁴. I, Quantification of explored area EA of α2 GABA_AR after 48 h of 4-AP application. Extra: t0 n = 2061 QDs, 48 h n = 546 QDs, p = 2.9 × 10⁻⁶. Syn; t0 n = 219 QDs, 48 h n = 74 QDs, p = 6.6 × 10⁻⁷. J, Quantification of α2 GABA_AR dwell time DT after 8 or 48 h of 4-AP application. Calculations were done for trapped or passing QDs at inhibitory synapses. Trapped: t0: n = 130 QDs, 8 h: n = 194 QDs, p = 0.007; t0: n = 85 QDs, 48 h: n = 51 QDs, p = 0.02; Passing: t0: n = 91 QDs, 8 h: n = 161 QDs, p < 0.001; t0: n = 91 QDs, 48 h: n = 31 QDs, p = 0.6. In B–D and J, data are presented as mean ± SEM. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 (Mann–Whitney rank sum test). In F–I, data are presented as median values ± 25%–75% IQR. *, p ≤ 0.05; ***, p ≤ 0.001 (Kolmogorov–Smirnov test). In all graphs, values were normalized to the corresponding control values.
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Figure 7.
GSK3β pathway influences gephyrin scaffold and GABA_ARs after changes in chronic activity. A, Morphology of neuron transfected with eGFP-S270A under control condition (t0) or in the presence of 4-AP after 8 or 48 h. Scale bar, 10 µm. B, Quantification of eGFP-S270A clusters after 8 or 48 h of 4-AP application. t0 n = 43 cells, 8 h n = 50 cells; 48 h n = 50 cells, 3 cultures. Cluster Nb: 0–8 h: p = 0.8, 0–48 h: p = 0.14; Area: 0–8 h: Mann–Whitney test p = 0.7, 0–48 h: p = 0.04; Intensity: 0–8 h: p = 0.12, 0–48 h: p < 0.001. C, Quantification of synaptic α2 GABA_AR clusters after 8 and 48 h of 4-AP compared with mock treated control. t0 n = 40 cells, 8 h n = 47 cells; t0: n = 59 cells, 48 h n = 52 cells, 3–5 cultures. Cluster Nb: 0–8 h: p = 0.8, 0–48 h: p = 0.7; Area: 0–8 h: p = 0.14, 0–48 h: p = 0.6; Intensity: 0–8 h: p = 0.03, 0–48 h: p = 0.4. D, Quantification of extrasynaptic α2 GABA_AR clusters after 8 and 48 h of 4-AP compared with mock treated control. Cluster Nb: 0–8 h: p < 0.001, 0–48 h: p = 0.7; Area: 0–8 h: p < 0.001, 0–48 h: p = 0.7; Intensity: 0–8 h: p < 0.001, 0–48 h: p = 0.3. E, Example traces of α2 GABA_AR trajectories at extrasynaptic (extra) and synaptic (syn) sites under control conditions (t0) or after 8 or 48 h of 4-AP application. Scale bar, 0.25 µm. F, Quantification of α2 GABA_AR diffusion coefficients after 8 h of 4-AP exposure. Extra: t0 n = 1580 QDs, 4AP 8 h n = 1892 QDs, p = 1.4 × 10⁻¹³. Syn: t0 n = 229 QDs, 8 h n = 307 QDs, p = 8.8 × 10⁻³; 3 cultures. G, Quantification of explored area EA of α2 GABA_AR after 8 h of 4-AP application. Extra: t0 n = 4575 QDs, 8 h n = 4041 QDs, p = 0.02. Syn: t0 n = 687 QDs, 8 h n = 663 QDs, p = 0.04. H, Quantification of α2 GABA_AR diffusion coefficients after 48 h of 4-AP exposure. Extra: t0 n = 314 QDs, 4AP 48 h n = 338 QDs, p = 0.05. Syn: t0 n = 46 QDs, 48 h n = 51 QDs, p = 0.04. 3 cultures. I, Quantification of explored area EA of α2 GABA_AR after 48 h of 4-AP application. Extra: t0 n = 939 QDs, 48 h n = 771 QDs, p = 0.02. Syn; t0 n = 138 QDs, 48 h n = 153 QDs, p = 0.04. J, Quantification of α2 GABA_AR dwell time DT after 8 or 48 h of 4-AP application. Calculations were done for trapped or passing QDs at inhibitory synapses. Trapped: t0: n = 82 QDs, 8 h: n = 97 QDs, p = 0.04; t0: n = 191 QDs, 48 h: n = 45 QDs, p = 0.5; Passing: t0: n = 104 QDs, 8 h: n = 131 QDs, p = 0.5; t0: n = 211 QDs, 48 h: n = 23 QDs, p = 0.1. In B–D and J, data are presented as mean ± SEM. *, p ≤ 0.05; ***, p ≤ 0.001 (Mann–Whitney rank sum test). In F–I, data are presented as median values ± 25%–75% IQR. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 (Kolmogorov–Smirnov test). In all graphs, values were normalized to the corresponding control values.
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Figure 8.
PKA, CAMKIIα, and GSK3β pathways are required to tune the inhibitory synapse. A, Quantifications of synaptic eGFP-SSA/S270 clusters and synaptic (α2 syn) and extrasynaptic (α2 extra) α2 GABA_AR clusters in relation to eGFP-WT show minor impact of the mutant under control condition. eGFP-WT n = 58 cells, eGFP-SSA/S270A n = 62 cells, 3 cultures. eGFP-SSA: Cluster Nb: p = 0.6; Area: p = 0.1; Intensity: p = 0.7. α2 syn: Cluster Nb: p = 0.001; Area: p = 0.1; Intensity: p = 0.02. α2 extra: Cluster Nb: p = 0.03; Area: p = 0.5; Intensity: p = 0.2. B, Quantification of median diffusion coefficient D of α2 GABA_AR in neurons expressing eGFP-WT or eGFP-SSA/S270A under control condition. Extra: WT n = 823 QDs, SSA/S270A n = 786 QDs, p = 0.004; Syn: WT n = 261 QDs, SSA/S270A n = 211 QDs, p = 0.3, 2 cultures. C, Quantification of α2 GABA_AR dwell time DT at synaptic sites in neurons expressing either eGFP-WT or eGFP-SSA/S270A. Calculations were done for all QDs (total), (trapped), or (passing) QDs at inhibitory synapses. No significant differences were found between eGFP-WT and eGFP-SSA/S270A. Total: WT n = 165 QDs, SSA/S270A n = 183 QDs, p = 0.1; Trapped: WT n = 95 QDs, SSA/S270A n = 116 QDs, p = 0.5; Passing: WT n = 70 QDs, SSA/S270A n = 67 QDs, p = 0.2. D, Quantification of eGFP-SSA/S270A clusters after 8 or 48 h of 4-AP application. t0 n = 53 cells, 8 h n = 45 cells, 48 h n = 51 cells, 3 cultures. Cluster Nb: 0–8 h: p = 0.3, 0–48 h: p < 0.001; Area: 0–8 h: p = 0.03, 0–48 h: p = 0.2; Intensity: 0–8 h: p = 0.3, 0–48 h: p = 0.9. E, Quantification of synaptic α2 GABA_AR clusters after 8 and 48 h of 4-AP compared with mock treated control. t0 n = 49 cells, 8 h n = 49 cells, 48 h n = 39 cells, 3 cultures. Cluster Nb: 0–8 h: p = 0.2, 0–48 h: p < 0.001; Area: 0–8 h: p = 0.8, 0–48 h: p = 0.6; Intensity: 0–8 h: p = 0.2, 0–48 h: p = 0.9. F, Quantification of extrasynaptic α2 GABA_AR clusters after 8 and 48 h of 4-AP compared with mock treated control. Cluster Nb: 0–8 h: p = 0.8, 0–48 h: p = 0.001; Area: 0–8 h: p < 0.001, 0–48 h: p = 0.7; Intensity: 0–8 h: p = 0.8, 0–48 h: p = 0.8. G, Quantification of α2 GABA_AR diffusion coefficients after 8 h of 4-AP exposure. Extra: t0 n = 624 QDs, 4AP 8 h n = 421 QDs, p = 5.4 × 10⁻⁷. Syn: t0 n = 252 QDs, 8 h n = 173 QDs, p = 0.2, 2 cultures. H, Quantification of explored area EA of α2 GABA_AR after 8 h of 4-AP application. Extra: t0 n = 1869 QDs, 8 h n = 1092 QDs, p = 7.8 × 10⁻¹⁴. Syn: t0 n = 753 QDs, 8 h n = 516 QDs, p = 0.07. I, Quantification of α2 GABA_AR diffusion coefficients after 48 h of 4-AP exposure. Extra: t0 n = 624 QDs, 4AP 48 h n = 631 QDs, p = 0.04. Syn: t0 n = 252 QDs, 48 h n = 251 QDs, p = 0.8. 2 cultures. J, Quantification of explored area EA of α2 GABA_AR after 48 h of 4-AP application. Extra: t0 n = 1092 QDs, 48 h n = 1890 QDs, p = 1.5 × 10⁻⁶. Syn; t0 n = 558 QDs, 48 h n = 750 QDs, p = 0.3. K, Quantification of α2 GABA_AR dwell time DT after 8 or 48 h of 4-AP application. Calculations were done for trapped or passing QDs at inhibitory synapses. Trapped: t0: n = 116 QDs, 8 h: n = 84 QDs, 48 h: n = 43 QDs, 0–8 h: p = 0.2; 0–48 h: p = 0.02; Passing: t0: n = 67 QDs, 8 h: n = 46 QDs, 48 h: n = 43 QDs, 0–8 h: p = 0.2; 0–48 h: p = 0.1. In A, C–F, and K, data are presented as mean ± SEM. *, p ≤ 0.05; ***, p ≤ 0.001 (Mann–Whitney rank sum test). In G–J, data are presented as median values ± 25%–75% IQR. *, p ≤ 0.05; ***, p ≤ 0.001 (Kolmogorov–Smirnov test). In all graphs except C, values were normalized to the corresponding control values.