Figure 8.

Presynaptic expression of α₂δ-2 induces upregulation of postsynaptic GABA_ARs in MSNs. A, B, Representative immunofluorescence micrographs of cultured MSNs cotransfected with distinct α₂δ subunits and soluble eGFP. Transfected neurons (21–28 DIV) were immunolabeled for vGAT and the GABA_AR. Colocalization of fluorescence signals within eGFP-filled axonal varicosities (arrowheads, axons are outlined with dashed lines) was analyzed using line scans. A, GABAergic synapses transfected with eGFP only (control) show matched presynaptic vGAT and postsynaptic GABA_AR immunoreactivity (summarized in sketch). B, Similar to control, postsynaptic GABA_ARs were localized opposite vGAT-positive presynaptic terminals expressing individual α₂δ isoforms (see also colocalization in line scans). Most importantly, GABA_AR clusters were larger and more intense opposite synaptic boutons expressing α₂δ-2 (blue arrowheads). C–F, Quantitative analysis of GABA_AR fluorescence intensity (C), cumulative frequency distribution of GABA_AR fluorescence intensity (D), bouton size (E), and vGAT fluorescence intensity (F). Values for individual cells (dots) and means (lines) ± SEM are shown. Values were normalized to the control within each culture preparation. Data from four independent culture preparations and 34–42 cells were analyzed in each condition. Statistics: ANOVA on log10-transformed data with Holm–Sidak post hoc analysis: C, F_(3,152) = 17.6, p < 0.001; post hoc: ***p < 0.001 between α₂δ-2 and control, ***p < 0.001 between α₂δ-2 and α₂δ-1/α₂δ-3, **p = 0.004 between control and α₂δ-3, p = 0.2 between control and α₂δ-1; E, F_(3,152) = 5.1, p < 0.01; post hoc: **p < 0.01 between α₂δ-3 and control/α₂δ-2, p = 0.08 between α₂δ-3 and α₂δ-1; F, F_(3,152) = 8.0, p < 0.001; post hoc: ***p < 0.001 between α₂δ-3 and control, **p < 0.01 between α₂δ-3 and α₂δ-1/α₂δ-2. Asterisks in graphs indicate the significant difference compared with control. Scale bars, 10 μm (A) and 3 μm (B).