FIGURE 2.

Activation of synaptic NMDARs increases surface expression of GABA_BRs. A, effects of glycine on GABA_B1, GABA_B2, and the AMPAR subunit GluA2 assessed by surface biotinylation. Cultured cortical neurons were treated with 200 μm glycine for 3 min and then incubated for 5 or 20 min without glycine before surface biotinylation and harvesting. Blots were probed with anti-GABA_B and anti-GluA2 antibodies and reprobed with anti-β-actin antibody to ensure equal loading and the specificity of surface biotinylation. B, quantification of the effect of chemLTP on GABA_B1 and GABA_B2. C, validation of chemLTP showing increased GluA2 protein surface expression. The results shown are the ratios of at least three independent experiments (n = 3). **, p ≤ 0.01 and ***, p ≤ 0.005 compared with control (Student's t test). D, GABA_BR endocytosis and recycling was measured by the loss of internalized GABA_BR specifically labeled with cleavable (S = S linked) biotin. Cortical cultures were surface-biotinylated as described above, and cells were activated by chemLTP protocol and incubated for 5 or 20 min to allow internalized receptors to recycle before cleavage of surface biotin. Residual biotinylated (internal) receptors were then isolated from cells by streptavidin pulldown, and GABA_BR subunits were detected by Western blotting. Blots were probed with anti- GABA_B1 and anti-GABA_B2 antibodies and reprobed with anti-β-actin antibody to ensure equal loading and the specificity of biotinylation. E, quantification of the effect of chemLTP on GABA_B1 and GABA_B2 rate of disappearance of biotinylated GABA_BRs provides a measure of receptor recycling. Leupeptin was included throughout the treatments to block any protein degradation or loss of internalized receptors. The results shown are the ratios of at least three independent experiments (n = 3). **, p ≤ 0.01 and ***, p ≤ 0.005 compared with control (Student's t test). § (p ≤ 0.005), significant differences between with and without AP5 treatments.