FIGURE 1.
Formation of synapse-like contacts between GABAergic MSNs and HEK293 cells expressing α1/β2/γ2-GABAARs at the cell surface. A, immunoblot analysis of the expression of GABAAR α1 (left panels) and β2 (right panels) subunits in a stable α1β2-HEK293 cell line in comparison with the normal HEK293 cells, using specific primary antibodies and alkaline phosphatase-conjugated secondary antibodies for detection. Representative blots of n = 2 independent experiments are shown. B, immunocytochemical analysis of the expression of GABAAR α1 (left panel) and β2 (middle panel) subunits in a stable α1β2-HEK293 cell line, using primary antibodies specifically binding to the extracellular epitopes of the respective subunits and Alexa488-coupled (left panel) or Alexa555-coupled (middle panel) secondary antibodies. Differential interference contrast (DIC) image of the same field (right panel) is included for comparison. Scale bar, 10 μm. Representative images of n = 2 independent experiments are shown. C, immunolabeling of synaptic contacts between GABAergic presynaptic terminals of MSNs expressing VGAT and α1β2γ2-HEK293 cells (top panel), α1β2-HEK293 cells (middle panel), or HEK293 cells (bottom panel). GABAergic terminals were labeled with an anti-VGAT antibody (in green); HEK293 cells were labeled with mCherry (in red), and the surface-expressed GABAARs were labeled with the γ2 subunit-specific antibody (in blue). A selected area in each image (left column, white box) was magnified four times, and these images were included in the right column. Scale bar, 10 μm. D, quantification of contacts between MSNs and α1β2γ2-HEK293 cells, α1β2-HEK293 cells, or control HEK293 cells expressed as a percentage of co-localized pixels between VGAT-positive MSN terminals and mCherry-expressing HEK293 cells. The data were first analyzed using Shapiro-Wilk and Kolmogorov-Smirnov tests and subsequently using the non-parametric Mann-Whitney test with a confidence interval of 95%. The box plots display the median and interquartile range (IQR); small squares represent the mean, and whiskers represent the data range within 1 S.D. of the median (n = 62–77 cells from n = 6 independent experiments). E, cell surface levels of GABAAR β2 subunit in α1β2-HEK293 cells showed no significant change following transient transfection of the γ2 subunit (α1β2γ2-HEK293 cells), as measured using ELISA with an extracellular epitope-specific anti-β2/3 subunit antibody, followed by an HRP-conjugated secondary antibody and colorimetric reaction (left panel; p > 0.05, paired t test). Total levels of GABAAR β2 subunit in α1β2γ2-HEK293 cells were reduced (right panel; p < 0.05, paired t test). The bar graphs represent the mean ± S.D. of the total of n = 4 independent experiments. The superimposed paired scatterplots represent the individual data points obtained from α1β2-HEK293 and α1β2γ2-HEK293 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001.