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.