Electrophysiology
We used standard intracellular recording technique (Thesleff, 1958; Santafé et al., 2003). Microelectrodes were prepared from borosilicate glass (World Precision Instruments, Sarasota, FL, USA) using a P97 micropipette puller (Sutter Instrument, Novato, CA, USA). Recording electrodes were 20-30 MΩ and filled with 3 M KCl. To record evoked and spontaneous (miniature) endplate potentials (EPPs and mEPPs, respectively) amplifier Axoclamp 900A and digitizer DigiData 1440A (Axon Instruments, San Jose, CA, USA) were used. Membrane potential was at -60 to -80 mV and recorded by miniDigi 1B (Axon Instruments, San Jose, CA, USA). The experiments with membrane potential deviations over 7 mV were declined. The nerve was stimulated with rectangular suprathreshold stimuli (0.2 ms duration, 0.5 Hz frequency) via a suction electrode connected to an isolated pulse stimulator, model 2100 (A-M Systems, Sequim, WA, USA). All electronic devices were driven by software pClamp v.10.4. Bandwidth was from 1 Hz to 10 kHz. After collecting 35 EPPs, mEPPs during 2 min were recorded. Quantal content estimated as ratio of averaged EPPs to mEPPs amplitude.