Pancuronium

The drugs, MS-222 (Pharmaq Ltd., UK), benzocaine and pancuronium (both from Sigma-Aldrich, France), were administered to the preparation using bath application of the respective substance in oxygenated Ringer solution at a rate of ∼2 ml/min, at a temperature of 17°C [17] (link). MS-222 at four different concentrations (0.0025, 0.005, 0.01 and 0.05%) and pancuronium (0.12 mg/ml) were dissolved in frog Ringer. In contrast, benzocaine was dissolved in 0.003% dimethylsulfoxide (DMSO, Sigma-Aldrich, France) before dilution in frog Ringer (final concentrations 0.0025, 0.005, 0.01 and 0.05%). All drug solutions were buffered to a pH 7.4 with NaHCO₃. A drug-related reduction of the spontaneous and evoked responses in similar isolated preparations usually occurred after 3–5 min and reached steady state after 15 min [20] (link). The wash-out of the different drugs was determined for a period of up to four hours in some experiments and revealed a dose- and drug-dependent recovery of the resting rate and stimulus-induced discharge modulation.

Zebrafish larvae at 5–6 dpf were paralyzed in 300 µM pancuronium (Sigma), immobilized in 2% low-melting-point agarose in a recording chamber, filled with embryo media and transferred to an electrophysiology platform. Local field potential (LFP) data were obtained from the optic tectum using a glass microelectrode. Electrodes were filled with 2 M NaCl, and electrical activity was recorded using an extracellular amplifier with a high impedance head stage (1700, A-M Systems). Signals were low-pass filtered at 5 kHz, high-pass filtered at 1 Hz, and digitized at 10 kHz using a digital acquisition board (Measurement Computing). Data were recorded and analyzed with DClamp (https://sites.google.com/site/dclampsoftware/home). As described in a previous study [14 (link)], ictal-like events were defined as electrical events greater than 5x baseline noise, multispike and > 500 ms in duration. Interictal-like events were defined as smaller discharges than ictal-like events with amplitudes greater than 3x baseline noise and > 100 ms in duration. Occurrences of electrographic epileptiform events (ictal-like events + interictal-like events) were analyzed.