Bicuculline

NG2DsRed mice received daily intraperitoneal (i.p.) injections (from P5-P11 or from P11-P15) of either 1 mg/kg bicuculline (abcam) in sunflower seed oil (Sigma 47123), 100 mg/kg vigabatrin in saline or 50 mg/kg tiagabine in saline (Tocris Biosciences). Control mice were injected with vehicle alone. Injection volume was 10 μl per g body weight, and solutions were warmed to 37 °C. The dose of bicuculline used was roughly half the CD₅₀ reported for P7 rats^{69 (link)}; two mice exhibited seizures and were excluded from the study.

The ubiquitination of AMPARs was induced by incubating neurons in artificial cerebrospinal fluid (ACSF; in mM, 25 HEPES, 120 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 30 D-glucose, pH 7.4) containing 40 μM bicuculline (Abcam) or 1 μM PMA (phorbol 12-myristate 13-acetate, Sigma) for 10 min at 37°C. In some experiments, neurons were pre-incubated with 1 μM PMA for 10 min prior to bicuculline treatment. For experiments involving the PKC blocker, neurons were pre-treated with 1 μM Go-6983 (Tocris) or DMSO (vehicle control) for 10 min prior to and present throughout the bicuculline or PMA/bicuculline treatments. Neurons were then lysed in warm 1% SDS (in PBS) and diluted in 10 volumes of ice-cold cell lysis buffer (1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM Na-pyrophosphate in PBS) supplemented with 10 mM NEM (N-ethylmaleimide, Sigma) and Complete EDTA-free protease inhibitor cocktails (Roche). Lysates were centrifuged at 14,000 rpm for 20 min at 4°C and cleared with protein A- or G-Sepharose beads. Pre-cleared lysates were then incubated with antibodies (anti-ubiquitin or anti-GFP) coupled to protein A- or G-Sepharose overnight at 4 °C, followed by four washes with ice-cold lysis buffer and elution in 2× SDS sample buffer. The immunoprecipitated proteins were resolved by SDS-PAGE and probed by western blot analysis with specific antibodies against GluA1, GluA2 and ubiquitin.

cLTP was performed on primary rat hippocampal and cortical neurons at DIV 15-17 as previously described, with some modifications (Tan et al., 2017 (link)). Briefly, neurons were washed and incubated in pre-warmed low Mg²⁺ artificial cerebrospinal fluid (ACSF; 120 mM NaCl, 2 mM CaCl₂, 5 mM KCl, 0.4 mM MgCl₂, 30 mM glucose, 25mM HEPES, pH 7.4) containing 0.5 μM tetrodotoxin (Abcam), 20 mM bicuculline (Abcam) and 5 μM strychnine (Sigma) at 37°C for 45 min. cLTP was induced by incubating neurons with low Mg²⁺ ACSF supplemented with 200 μM glycine, 20 μM bicuculline and 5 μM strychnine at room temperature for 5 min. To inhibit the activity of CaMKIIα, neurons were treated with KN-93 (Abcam, 10 μM) for 15 min prior to and during cLTP induction.