Tetracaine hydrochloride

CF-1 mice were kindled according to the optimized protocol defined by Matagne and Klitgaard (67 (link)) and Rowley and White (68 (link)). Briefly, mice were stimulated twice daily (5 days/week) with a corneal stimulation of 3 mA (60 Hz) for 3 s. Prior to each stimulation, a drop of 0.9% saline containing 0.5% tetracaine hydrochloride (Sigma-Aldrich, St. Louis, MO, United States) was applied to the cornea to ensure local anesthesia and good electrical conductivity. Stimulations were delivered 4 h apart. Animals were considered kindled when they displayed five consecutive stage five seizures according to the Racine scale (69 (link), 70 (link)).

One minute before stimulation, a drop of ocular anesthetic (1% solution of tetracaine hydrochloride; Sigma-Aldrich, St. Louis, MO, USA) was applied to each eye of the mice. The maximal electroshock seizures were induced by constant current stimuli (50 Hz sinewave, 0.2 s) using a rodent shocker (type 221; Hugo Sachs Elektronik, Freiburg, Germany). An alternating sinusoidal current was administered through transcorneal electrodes, soaked in saline. During stimulation, mice were manually immobilized by the hand of the experimenter for 3–5 s. Immediately after stimulation, animals were placed in a Plexiglas arena for behavioral observation. The endpoint was a tonic extension of the hindlimb that exceeded a 90° angle with the body. The thresholds for maximal electroconvulsion were assessed by the “up-and-down” method described by Kimball et al. [75 (link)]. The current intensity was lowered or raised by 0.06-log intervals, depending on whether the previously stimulated animal did or did not exert tonic hindlimb extension, respectively [74 ]. Each mouse was stimulated only once at any given current intensity. The data obtained in groups of 20 animals were used to determine the threshold current-causing endpoint in 50% of the mice (CS₅₀ with confidence limits for 95% probability).

Tetracaine hydrochloride and lidocaine hydrochloride monohydrate were obtained from Merck KGaA (Darmstadt, Germany). Tetrodotoxin standard was purchased from Tocris Bioscience (Bristol, UK) and the standard solution was prepared at 1 mg/mL in 1% acetic acid. The automated patch clamp Patchliner with 8 amplifier channels, 2 HEKA EPC10 Quadro amplifiers, NPC-16 borosilicate recording chips (medium resistance), external solution (140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM D-glucose monohydrate, 10 mM HEPES/NaOH, pH 7.4), internal solution (50 mM CsCl, 10 mM NaCl, 60 mM CsF, 20 mM EGTA, 10 mM HEPES/CsOH, pH 7.2), and seal enhancer solution (10 mM HEPES, 130 mM NaCl, 5 mM glucose, 4 mM KCl, 10 mM CaCl₂, 1 mM MgCl₂, pH 7.4, MOsm 302) were obtained from Nanion Technologies GmbH (Munich, Germany). Neuroblastoma murine (Neuro-2a) cells were purchased from ATCC LGC standards (Manassas, VA, USA). Foetal bovine serum (FBS), penicillin/streptomycin solution, phosphate-buffered saline (PBS), Roswell Park Memorial Institute (RPMI) medium, sodium pyruvate, trypsin-EDTA enzyme, ouabain, veratridine, and thiazolyl blue tetrazolium bromide (MTT) were purchased from Merck KGaA (Darmstadt, Germany).