Whole-cell patch clamp recordings

Neurons were recorded at 31 °C under superfusion with artificial cerebrospinal fluid containing (in mM) 125 NaCl, 20 D-glucose, 10 HEPES, 4 MgCl₂, 2 KCl, 1 CaCl₂ (pH = 7.4). Whole-cell recordings measuring GABABR currents were made with glass pipettes containing the following internal solution (in mM): 110 K-methylsulfonate, 20 KCl, 10 HEPES, 10 EGTA, 4 MgATP, 0.4 Na₃GTP, 10 Na phosphocreatine, 1.8 MgCl₂. Recordings were made in the presence of TTX (1 μM), kynurenate (1 mM), and R,S-MCGP (500 μM), and bicuculline (20 µM). In current-clamp configuration the resting membrane potential (Vm) was monitored for 5 min to ensure a stable baseline, followed by the addition of bath applied baclofen (20 µM). Once Vm reached a steady-state in the baclofen treatment, the drug was either washed out or the GABABR antagonist {"type":"entrez-protein","attrs":{"text":"CGP52132","term_id":"877594719","term_text":"CGP52132"}}CGP52132 (20 µM) was added to the bath. The Vm was monitored until it reached a steady-state. Whole-cell patch clamp recordings were performed with borosilicate glass micropipettes filled with either (in mM) 135 CsCl, 10 HEPES, 10 EGTA, 4 MgATP and 0.4 Na₃GTP (pH = 7.4) for recording tonic, GABAAR-mediated currents or 105 CsMeSO₄, 10 CsCl, 10 HEPES, 10 EGTA, 4 MgATP, and 0.4 Na₃GTP for recording muscimol-evoked currents. Cells were held at −70 mV. GABAAR-mediated currents were recorded in the presence of TTX (1 µM), NBQX (10 µM) and D,L-APV (100 µM). Access and input resistance were regularly monitored with −5 mV voltage steps. All recordings were made using an Axopatch 200B amplifier (Molecular Devices), filtered at 2 kHz and digitized at 25 kHz. All the data were collected using the Clampex 10 program and analyzed using Clampfit 10 (Axon). Currents were analyzed offline using Clampfit software.