Fig. 1.

(A) Cortical neurons were left untreated or exposed to 20 μM glutamate for 0–30 min. Biotinylated (surface) and total proteins were visualized by immunoblotting with GABA_BR2 (R2) and GABA_BR1 (R1) antibodies. (Lower) Immunoblots for each condition were analyzed by densitometry. **P < 0.01. (B) Surface expression of GABA_BR2 was evaluated as above after exposure to 20 μM NMDA for 0–10 min. (Lower) Immunoblots were quantified as above. *P < 0.05; **P < 0.01. (C) Whole-cell recordings from cultured hippocampal neurons with 10 μM baclofen-activated K⁺ currents before (Left) and after (Right) glutamate receptor activation (1 mM, 15 s) in the absence (Upper) and presence (Lower) of 100 nM MK801 at 7, 10, 13, and 19 min. Baclofen responses were obtained in a high K⁺ Krebs’ solution supplemented with AP-5 (20 μM), CNQX (10 μM), TTX (500 nM), and Bicuculline (25 μM). (D) Baclofen-activated currents normalized to the first response at t = 0 min were recorded as control responses (■) and responses interposed with glutamate (○) or glutamate and MK801 applications (▲). All points represent the mean ± SEM (n = 5–12 cells; *P < 0.05). (E) Cortical neurons were left untreated or exposed to 20 μM glutamate, 20 μM PAO, or 20 μM glutamate plus 20 μM PAO for 30 min. Biotinylated (surface) and total proteins were visualized by immunoblotting with GABA_BR2 and GABA_BR1 antibodies. (F) Hippocampal neurons were transfected with MYC-GABA_BR1a and HA-GABA_BR2 and processed for immunoendocytosis with MYC antibodies. Cells were left untreated (Upper) or exposed to 20 μM glutamate for 30 min (Lower). Surface receptors were detected with MYC- and Texas Red-conjugated secondary antibodies (red channel). Internalized receptors were detected using FITC-conjugated secondary antibodies (green channel). (Right) Merged images are shown. (Scale bar: 5 μm.)