Figure 4.

Plasma membrane insertion of GABA_A receptors is increased by vigabatrin treatment. Low-density cultures were incubated in the absence (control) or presence of VGB (0.5 mg/ml, 37°C) for 18 h. Anti-β2/3 subunit antibody immunofluorescence labeling in living cells was performed at room temperature (permissive for receptor insertion but not for endocytosis). Immunofluorescence was visualized by confocal microscopy. A Z-series of ten optical sections, each 0.2 μm thick, was collected and a 3D reconstruction was rendered. (A) Representative image. (B) Immunofluorescence intensity, puncta size and puncta number was quantified by an investigator blinded to the experimental conditions. Data shown are average ± SEM, n = 7. Comparisons between control and VGB treatment groups were made using paired t-tests corrected for multiple (3) comparisons *p ≤ 0.01. (C) Control experiment showing that receptor endocytosis occurs at 37°C but not room temperature. Living neurons were incubated in anti-β2/3 subunit antibody at either room temperature or 37°C to measure receptor endocytosis. After this incubation period the surface receptor population was labeled with Alexa 488-conjugated secondary antibody (green). Cells were then fixed, permeabilized and the endocytosed receptors were labeled with Alexa 594-conjugated secondary antibody (red). The brightness and contrast settings of images for receptor endocytosis (red) were enhanced to show that not even a faint signal is detectable at room temperature.