Description

The action potential (AP) is the basic signaling unit in various crucial physiological processing, for instance, in neurotransmission, muscle contraction, and glandular secretion (Koch, 1990). The classic model animal, Caenorhabditis elegans (or C. elegans), with a simple and compact nervous system, conservatively employs the calcium-mediated all-or-none APs for odor response in AWA olfactory neurons (Liu et al., 2018), as well as for muscle contraction in body wall muscles (Gao and Zhen, 2011; Liu et al., 2011) and pharyngeal muscles (Davis et al., 1999). Plateau potentials were also observed in ASE and RMD neurons (Goodman et al., 1998; Mellem et al., 2008; Lockery et al., 2009; Lockery and Goodman, 2009), though the underlying roles in specific behavior are still elusive. Either in neurons or in muscles, the action potential firing is dependent on the excitatory pre-synaptic vesicles release. The minimum number of the presynaptic vesicles to elicit a single action potential in C. elegans has not been reported before. Here, by the combination of optogenetics with in-vivo patch clamping technology, we demonstrated that at least approximately 37 excitatory acetylcholinergic vesicles are required for the initiation of an action potential at post-synaptic body wall muscles.

We used the transgenic strain ZX460 with zxIs6 that expresses the optogenetic protein, ChR2 (H134R), in all excitatory cholinergic motor neurons. By gradually increasing the irradiated blue light intensity (from 1-10% of the full intensity 8 mW/mm²), graded electrical responses and eventually all-or-none APs were evoked at the neuromuscular junction. Graded increased excitatory postsynaptic currents (EPSCs) were also recorded. The used holding potential of -30 mV is the reversal potential of postsynaptic ionotropic GABA receptor and therefore the GABAergic inhibitory PSCs could not be recorded (Gao and Zhen, 2011; Maro et al., 2015). Specifically, as the light intensity reached 8% of the full intensity (~0.64 mW/mm²), solid action potentials could be evoked (Fig 1A). Consistent with the all-or-none principle, the amplitude of the evoked muscular action potentials was no longer changed even with stronger light stimulation (Fig 1B, bc). The AP half-width, however, was slightly shortened and was correlated with higher EPSCs charge transfer (Fig 1C). The average smallest EPSCs quantity of electric charge is -1.217 pC for the elicitation of an action potential in muscle cells. To evaluate the single vesicle charge transfer, we measured miniature-EPSCs holding at -30 mV (Fig 1D). The average miniature-EPSCs quantity of electric charge is -32.7 fC (Fig 1E), thus the calculated minimum number of cholinergic vesicles is 37 by dividing the quanta charge into the smallest EPSCs charge.