Introduction
Acetylcholine (ACh) is the main neurotransmitter in the peripheral nervous system of vertebrates and humans. In particular, it is responsible for the transmission of signals from the motor nerve to skeletal muscle (Del Castillo & Katz, 1957; Ciani & Edwards, 1963). Since neuromuscular junction (NMJ) is a key linker in the initiation of any motor act (from voluntary movement of the limbs to breathing and contraction of the vocal cords), investigation of the regulation of neuromuscular transmission is of great importance for both fundamental neurobiology and applied medicine.
Since the midst of the 20th century, the data began to accumulate indicating that ACh, released in the synaptic cleft from the nerve endings, activates presynaptic cholinergic receptors, thus exerting a modulatory effect on the neurotransmission process by changing the amount and/or dynamics of subsequent portions of neurotransmitter release (Ciani & Edwards, 1963; Starke et al., 1989; Bowman et al., 1990; Prior et al., 1995; Miller, 1998; Nikolsky et al., 2004). Initially pharmacologically, and later by other methods it has been shown that both ionotropic nicotinic and metabotropic muscarinic cholinergic receptors are present in the motor nerve terminal, and activation of these receptors can lead to autoregulation of ACh release (Bowman et al., 1990; Miller, 1990; Santafé et al., 2004).
When studying autoregulation mediated by muscarinic cholinergic receptors, it was found that activation of the M1-subtype receptors led to facilitation of the release. In contrast, activation of the M2-subtype caused inhibition of the ACh quanta release (Oliveira et al., 2002; Santafé et al., 2003). Both M1‐ and M2‐mediated mechanisms depend on calcium influx (Santafé et al., 2003; Slutsky et al., 2003; Khaziev et al., 2016; Zhilyakov et al., 2019).
Studies of the mechanisms of autoregulation of ACh release mediated by nicotinic cholinergic receptors are complicated by the fact that the predominant population of these proteins is located in the postsynaptic membrane. Their activation is accompanied by depolarization of sarcolemma and subsequent generation of action potential, which ultimately leads to muscle contraction. The data collected by to date indicate that activation of presynaptic nicotinic cholinergic receptors leads to inhibition of the process of ACh release (Van der Kloot, 1993; Prior & Singh, 2000; Balezina et al., 2006).
Also, experimental evidence was obtained indicating possible involvement of voltage-gated calcium channels (VGCCs) of L-type (Cav1) in modulation of neurotransmission (Prior & Singh, 2000). Meanwhile, the results of a number of studies demonstrate that neither the Cav1 type nor the N-type (Cav2.2) VGCCs participates in the evoked release of ACh in mammalians mature neuromuscular contacts (Penner & Dreyer, 1986; Atchison, 1989; Protti et al., 1991; Bowersox et al., 1995).
Thus, the question on the role of calcium channels in the mechanisms of regulation of ACh release, mediated by nicotinic cholinergic receptors, remains open as of now.
In the present study, using a pharmacological approach, electrophysiological techniques and the method of optical registration of changes in the calcium level in the motor nerve ending, we made the following observations. An agonist of nicotinic receptors (at a concentration not significantly affecting the state of the postsynaptic membrane) leads to a decrease in the amount of released ACh quanta. This effect is accompanied not by a decrease, but by an increase of calcium ions entry into the motor nerve terminal. Our data suggest that nicotinic cholinergic receptors responsible for the mechanism of ACh release autoregulation are the receptors of neuronal type. Activation of these receptors leads to upregulation of Cav1 type of VGCCs, resulting the enhancement of Ca2+ entry into the nerve ending.