Xe991

Tertiapin-Q (STT-170, Alomone Labs), CGP54626 (1088, Tocris), baclofen (0417, Tocris), linopirdine (1999, Tocris), XE991 (2000, Tocris), PK-THPP (5338, Tocris), spadin (5594, Tocris), and tolbutamide (T0891, dissolution with alcohol, Sigma) were used in whole-cell patch clamp mode. Kynurenic acid (K3375, Sigma), picrotoxin (1128, Tocris), and tetrodotoxin (T-550, Alomone Labs) were used to block synaptic currents. All solutions used in this study were made according to manufacturer’s specifications, and stock solutions of all drugs were dissolved in autoclaved de-ionized water unless specifically stated.

Drugs were bath applied at the following final concentrations: 10 µM 6-cyano-7-nitoquinoxaline-2,3-dione (CNQX; AMPA-type glutamate receptor antagonist), 100 µM d-2-amino-5- phosphonovaleric acid (APV; NMDA-type glutamate receptor antagonist), 100 µM picrotoxin (PTX; GABA-A receptor blocker), 1 µM tetrodotoxin (TTX; voltage-dependent Na⁺ channel blocker), 10 µM XE991 (KCNQ channel blocker), 4.0 mM CsCl. Drugs were obtained from Sigma, except for XE991 and ZD7288, purchased to Tocris, and TTX, that was obtained from Alomone Labs. The synaptic blockers were present in the pharmacological experiments performed to identify ionic currents involved in resonance.

The neurons were filled with Alexa 594 (20 μM, Life Technologies) through a recording pipette, and the drugs were applied with another glass pipette under an observation with a two-photon microscope (FV1000MPE, Olympus). The pipette contained Alexa 594 (20 μM) and either DTX (400 nM, Alomone) or XE991 (200 μM, Tocris), and it was placed on the axon at 14.0±0.9 μm (n=4) and 17.8±0.7 μm (n=6) away from the soma for control and deprived neurons, respectively. The iontophoretic pulse (20 ms) was applied before a somatic depolarization with a 10-ms separation. The effects disappeared when the pipette did not include the drugs, or it was displaced from the AIS.

For current clamp and calcium imaging recordings drugs were administered into the bath saline. Substances used were then added to the superfusate from thawed stock solutions. Muscarinic toxin mamba toxin 7 (MT-7) and the KCNQ agonist retigabine were obtained from Peptides International (Cat. number PMT-4340-s, Louisville, KY, USA). Muscarine, bicuculline, and biocytin were obtained from Sigma-Aldrich-RBI (St. Louis, MO, USA). KCNQ antagonist XE991 was obtained from Tocris (Bristol, UK).

A hydrogen water generator from Hydrogen (Osmo-star Soriano S.L., Alicante, Spain) was employed to prepare HRW, as described by [27 (link)]. The XE-991, acquired from Tocris Bioscience (Ellisville, MO, USA), SnPP from Frontier Scientific (Livchem GmbH & Co., Frankfurt, Germany), ML-385 and dicoumarol from Eurodiagnostico S.L. (Madrid, Spain) were dissolved in dimethyl sulfoxide (1% solution in SS).
All of the drugs were intraperitoneally administered to a final volume of 10 ml/kg. HRW at 0.3 mM was administered 1 h before testing, whereas 12 µmols/kg of XE-991, 25 mg/kg of ML-385, or 10 mg/kg of SnPP or dicoumarol were administered 30 min before the tests, in conformity with another study [27 (link)]. All of the drugs were newly prepared before administration. For each group treated with a drug, the respective control group received the same volume of analogous vehicles.

Drugs were bath-applied at the following final concentrations: 10 μM 6-cyano-7-nitoquinoxaline-2,3-dione (CNQX; AMPA-type glutamate receptor antagonist), 100 μM d-2-amino-5- phosphonovaleric acid (APV; NMDA-type glutamate receptor antagonist), 100 μM picrotoxin (PTX; GABA_A receptor blocker), 1 μM tetrodotoxin (TTX; voltage-dependentNa⁺ channel blocker), 10 μM XE991 (KCNQ channel blocker), 60 μM phenytoin (PHT, I_NaP blocker). Drugs were obtained from Sigma, except for XE991 purchased from Tocris, and TTX that was obtained from Alomone Labs.

A-ITC and P-ITC, obtained from Sigma-Aldrich (St. Louis, MO, USA) and XE-991, purchased in Tocris Bioscience (Ellisville, MO, USA) were dissolved in SS. All drugs were freshly prepared before use and intraperitoneally administered in a final volume of 10 mL/kg, 30 min, and 45 min before testing, in accordance with our preliminary studies and other work [12 (link)]. For each group treated with a drug, the respective control group received the same volume of vehicle.