6 cyano 7 nitroquinoxaline 2 3 dione cnqx

The artificial cerebrospinal fluid (ACSF) consisted of (in mM) 125 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, 2.5 KCl, 10 glucose and was equilibrated with 95% O₂ / 5% CO₂ at least 1 h before use (pH 7.4, osmolarity 306 mOsm). The slices for the PV-reporter mice were cut in a choline chloride-based solution consisting of (in mM) 37.5 (choline chloride, 87 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 0.5 CaCl₂, 7 MgCl₂, 25 NaHCO₃ and 25 d-glucose). The pipette solution was composed of (in mM) 128 K-gluconate, 2 KCl, 4 NaCl, 1 CaCl₂, 11 EGTA, 10 K-HEPES, 2 Mg₂-ATP, 0.5 Na-GTP and 2 lidocaine-N-ethyl chloride (pH adjusted to 7.4 with KOH and osmolarity to 306 mOsm with sucrose). Dimethylsulfoxide (DMSO) and lidocaine-N-ethyl chloride were obtained from Sigma-Aldrich (St. Louis, Missouri); gabazine (SR95531), DL-2-Amino-5-phosphonopentanoic acid (APV) and 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) were obtained from Biotrend (Cologne, Germany). APV, CNQX and gabazine were used from stock solutions in DMSO. The DMSO concentration of the final solution never exceeded 0.1%.

The bathing solution consisted of (in mM) 125 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₅, 1 MgCl₂, 2 CaCl₂, 2.5 KCl, 10 glucose and was equilibrated with 95% O₂/5% CO₂ at least 1 h before use (pH 7.4, osmolarity 306 mOsm). Two pipette solutions for whole-cell recordings were used: for a pipette Cl^- concentration ([Cl^-]_p) of 10 mM the solution was composed of (in mM) 128 K-gluconate, 2 KCl, 4 NaCl, 1 CaCl₂, 11 EGTA, 10 K-HEPES, 2 Mg²-ATP, 0.5 Na-GTP, and 2 lidocaine-N-ethyl chloride (pH adjusted to 7.4 with KOH and osmolarity to 306 mOsm with sucrose). For a [Cl^-]_p of 50 mM the solution contained (in mM) 86 K-gluconate, 44 KCl, 4 NaCl, 1 CaCl₂, 11 EGTA, 10 K-HEPES, 2 Mg2-ATP, 0.5 Na-GTP. Pipette solution for gramicidin-perforated patch experiments consisted of (in mM) 10 Na-gluconate, 120 KCl, 1 CaCl₂, 2 MgCl₂, 11 EGTA, and 10 K-HEPES. For gramicidin-perforated patch-clamp recordings 10 μg/ml Gramicidin D (Sigma, St. Louis, MO, United States) was added from a stock solution (2 mg/ml in DMSO) on the day of experiment. Gramicidin D, bumetanide, and lidocaine-N-ethyl chloride was obtained from Sigma-Aldrich and 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) was obtained from Biotrend (Cologne, Germany). CNQX and bumetanide were used from a dimethylsulfoxide (DMSO, Sigma-Aldrich) stock solution. The DMSO concentration of the final solution never exceeded 0.1%.

Patch-clamp pipettes (4–6 Mohm resistance) were prepared from borosilicate glass (BF150-86-10; Harvard Apparatus, Holliston, MA, USA) using a DMZ pipette puller (Zeitz). Patch-clamp recordings were performed using an EPC-10 amplifier (HEKA Elecktronik GmbH, Reutlingen, Germany) with the following intracellular solution (in mM): 105 K-gluconate, 10 HEPES, 10 phosphocreatine-Na, 4 ATP-Na2, 30 KCl (pH 7.25, adjusted with KOH). Intrinsic excitability was measured in current-clamp using depolarizing current steps of increasing amplitude (0–500 pA, 500 ms) at an interstimulus interval of 6 s, from a cell potential set to −70 mV, in the presence of SR95531 hydrobromide (Gabazine, 10 μm, Hello Bio) to block GABA_A receptor-mediated synaptic transmission, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μm, Biotrend, Köln, Germany) to block AMPA receptor-mediated synaptic transmission and D-2-amino-5-phosphonopentanoic acid (D-APV, 50 μm, Hello Bio) to block NMDA receptor-mediated synaptic transmission.