Experiments were performed on isolated nerve-muscle preparations of musculus cutaneous pectoris from the frog Rana ridibunda. The experimental procedures were performed in accordance with the guidelines for use of laboratory animals of Kazan Federal University and Kazan Medical University, in compliance with the NIH Guide for Care and Use of Laboratory Animals. Experimental protocols met the requirements of the European Communities Council Directive 86/609/EEC and were approved by the Ethical Committee of Kazan Medical University.

Cytosolic concentration of ionized Ca²⁺ ([Ca²⁺]_i) in nerve endings was monitored using fluorescent microfluorimetry (Tsien, 1989). Each nerve-muscle preparation was loaded with Ca²⁺-sensitive dye by soaking nerve stump in 50 mM solution of fluorescent Ca²⁺-indicator Oregon Green 488 BAPTA-1 Hexapotassium Salt (Molecular Probes, Eugene, Oregon, USA); for details of loading technique see (Peng and Zucker, 1993; Wu and Betz, 1996; Tsang et al., 2000; Samigullin et al., 2015). At the end of loading protocol, all terminals in the proximal part of the nerve trunk had sufficient levels of fluorescence. It has been estimated that the intra-terminal concentration of the probe varied between 40 and 150 μM (Suzuki et al., 2000).

The fluorescent probe by definition binds Ca²⁺ and hence may affect both [Ca²⁺]_i dynamics and physiology of the neuromuscular junction. Our own observations (Samigullin et al., 2015) as well as observations of others (Wu and Betz, 1996) failed to detect any appreciable influence of loaded Ca²⁺ probe on the amplitude of the postsynaptic response or on the frequency of the miniature end-plate potentials. Nonetheless we additionally performed control experiments to compare spontaneous endplate currents (mEPC) and quantal release before and after Ca²⁺ probe loading. It appeared that Ca²⁺ probe affects neither; the mEPC frequency was 0.32 ± 0.03 Hz (n = 4, P < 0.05) before and 0.32 ± 0.15 Hz (n = 4, P < 0.05) after loading of the probe; similarly the quantal content was 1.06 ± 0.09 (n = 3, P < 0.05) and 0.99 ± 0.07 (n = 3, P < 0.05). We may conclude, therefore adding Ca²⁺ probe into the cytosol of the terminal does not affect physiological parameters of neurotransmission.

Neuromuscular preparations were continuously perfused with the Ringer solution of the following content (in mM): NaCl–113, KCl–2.5, NaHCO₃–3, MgCl₂–6, CaCl₂–0.9; pH was adjusted to 7.4. Low extracellular Ca²⁺ (0.9 mM) and high level of external MgCl₂ (6 mM) were used to block muscle contraction. All experiments were performed under these conditions except the serie with decreasing extracellular Ca²⁺. Experiments were performed at 20.0 ± 0.3°C.

Fluorescent signal was recorded using photometric setup on the base of Olympus BX-51 microscope with x60 water-immersion objective connected to photodiode S1087 (Hamamatsu, Japan) as described in Sinha and Saggau (1999) and Samigullin et al. (2010, 2015). The region for recording was selected by optical viewfinder (Till Photonics, Munich, Germany). Excitation light (488 nm) was generated by Polychrome V (Till Photonics, Munich, Germany). To minimize bleaching of the dye and decrease background fluorescence, the recording area of nerve terminal was restricted by an iris diaphragm. Illumination was controlled by a shutter with a typical exposure time of 400 ms and a delivery rate of 0.5 Hz. Motor nerve was electrically stimulated by rectangular voltage pulses of 0.2 ms in duration and supra-threshold amplitude at a frequency of 0.5 Hz using the “suction” electrode described earlier (Kazakov et al., 2015). The photodiode signal was digitized by the ADC Digidata 1440A (Molecular Devices, USA) with sampling rate 10.256 kHz. Fluorescence recordings, illumination and electrical stimulation were all controlled by WinWCP software (Strathclyde University, Glasgow, UK). The peak amplitude of [Ca²⁺]_i transients was measured and changes in fluorescence are represented as ΔF/F₀ (the change in fluorescence intensity relative to the background fluorescence as a percentage). For each experiment, about 60 fluorescence responses were averaged.

Spontaneous and evoked endplate currents (mEPC and EPC, respectively) were recorded with a two-electrode voltage clamp technique at a holding potential of −60 mV. Intracellular microelectrodes (5–10 MΩ in resistance) were filled with 2.5 M KCl. Currents were recorded using Axoclamp 900A amplifier and digitized by Digidata 1440A (Molecular Devices, USA) under control of Clampex software v. 10.5. Quantum content of EPCs was calculated by dividing the area under EPC curve by the area under mEPC curve.

All reagents were obtained from Sigma (Saint Louis, Missouri, USA). Drugs were diluted in extracellular solution to get the following final concentrations: carbachol (10 μM), acetylcholine (100 μM), neostigmine (1 μM), atropine (1 μM), d-tubocurarine (10 μM), muscarine (10 μM), nicotine (10 μM), pirenzepine (100 nM), methoctramine (10 nM), mecamylamine (640 nM–6.9 μM), metillikakonitin (10 nM), ω-conotoxin GVIA (300 nM).

Experimentally measured relative amplitudes were tested for normal distribution. Statistical significance of relative amplitudes we assessed by Student's t-test for pairwise variant. Then data are presented as mean (%) ± SEM. Values of P < 0.05 were considered significant.

Application of 10 μM of carbachol decreased amplitudes of mEPC and EPC by 26 ± 5% (n = 4, P < 0.05) and 61 ± 8% (n = 4, P < 0.05), respectively (Figure 1A). Carbachol also reduced quantal content by 46 ± 10% (n = 4, P < 0.05) as compared to control (Figure 1B). 100 μM ACh attenuated quantal content by 49 ± 7% (n = 18, P < 0.05, Figure 1B). Both carbachol and ACh also attenuated amplitude of [Ca²⁺]_i transients by 11 ± 1% (n = 5, P < 0.05) and 13 ± 4% (n = 6, P < 0.05,) respectively (Figures 1C,D). Since inhibitory effects of exogenous acetylcholine and carbachol were similar, we used carbachol in all subsequent experiments to activate nicotinic and muscarinic receptors simultaneously. Nicotine and muscarine, however, were used to selectively activate respective receptors types. Also, carbachol is not hydrolysed by acetylcholine esterase, and hence its concentration remains constant over the entire experiment duration.

We were interested to elucidate whether a 11% decrease in the peak magnitude of [Ca²⁺]_i transient is sufficient to account for a 46% decline in neurotransmitter release observed with carbachol (Figures 1A–C). For this purpose, we have reduced the external concentration of Ca²⁺ from 0.6 to 0.3 mM and found that it decreased the evoked [Ca²⁺]_i transient by 23 ± 4% (n = 3) (Figure 2A). In parallel with this, quantal content dropped by 80 ± 4% (Figure 2B). Thus, carbachol-induced decrease in the size of evoked [Ca²⁺]_i transients is more than sufficient to produce inhibition of neurotransmitter release observed in our experiments earlier. Similar observations were made previously while studying quantal content and [Ca²⁺]_i transients under activation of cannabinoid receptors (Newman et al., 2007).

Exposure to 10 μM nicotine reduced the amplitude of [Ca²⁺]_i transient by 15 ± 3% (n = 6, P < 0.05, Figure 3B). Application of 10 μM muscarine decreased the amplitude of [Ca²⁺]_i transient by 8 ± 2% (n = 5, P < 0.05, Figure 3B). Nicotinic antagonist d-tubocurarine (10 μM) partially but significantly reduced nicotine effect on the [Ca²⁺]_i transient, while incubation with muscarinic blocker atropine (1 μM) completely eliminated presynaptic effect of muscarine (Figure 3B). In the presence of atropine (1 μM), carbachol (10 μM) reduced [Ca²⁺]_i transient by 9 ± 5% (n = 6, P < 0.05, Figure 3B), while in the presence of d-tubocurarine (10 μM)—by 18 ± 9% (n = 4, P < 0.05, Figure 3B). Pre-incubation with the mixture of d-tubocurarine (10 μM) and atropine (1 μM), completely eliminated carbachol effects on [Ca²⁺]_i transients (Figures 3A,B).

Nicotinic blocker d-tubocurarine did not change the inhibitory effect of muscarine alone—it reduced [Ca²⁺]_i transients by 8 ± 2% (n = 7, P < 0.05, Figures 4A,B). Similarly, blockade of muscarinic receptors by atropine did not significantly affect the inhibitory action of nicotine –[Ca²⁺]_i transients were reduced by 8 ± 3% (n = 7, P < 0.05, Figures 4C,D).

M₁-receptor blocker pirenzepine (100 nM) by itself decreased [Ca²⁺]_i transients by 14 ± 7% (n = 5, P < 0.05, Figures 5A,B). Addition of muscarine further decreased the amplitude of [Ca²⁺]_i transient (Figures 5A,B). Exposure to 10 nM methoctramine (specific blocker of muscarinic M₂ receptor subtypes), did not affect [Ca²⁺]_i transients by itself. In the presence of methoctramine, however, the inhibitory action of muscarine on [Ca²⁺]_i transient was completely eliminated (Figures 5C,D).

In these experiments nicotine was used as a specific agonist of nicotinic acetylcholine receptors. Mecamylamine is able to block various subtypes of nicotinic receptors depending on concentration within the range from 640 nM to 6.9 μM (Papke et al., 2001; Rabenstein et al., 2006; Ostroumov et al., 2008). Next, we tested the effects of different concentrations of mecamylamine. Application of 640 nM mecamylamine increased [Ca²⁺]_i transient amplitude by 18 ± 3% (n = 5, P < 0.05, Figure 6). When 640 nM mecamylamine was applied with nicotine [Ca²⁺]_i transient amplitude decreased by 9 ± 4% (n = 5, P < 0.05, Figure 6). At 2.5 μM, mecamylamine increased [Ca²⁺]_i transient amplitude by 16 ± 2% (n = 3, P < 0.05, Figure 6), while subsequent addition of nicotine reduced [Ca²⁺]_i transient amplitude by 19 ± 4% (n = 3, P < 0.05, Figure 6). At 3.6 μM mecamylamine increased [Ca²⁺]_i transient by 23 ± 7% (n = 6, P < 0.05, Figure 6), whereas its combined application with nicotine decreased [Ca²⁺]_i transient by 14 ± 2% (n = 6, P < 0.05, Figure 6). Finally, at 6.9 μM, mecamylamine increased [Ca²⁺]_i transient by 15 ± 8% (n = 3, P < 0.05, Figure 6), while combined application with nicotine decreased [Ca²⁺]_i transient amplitude by 8 ± 2% (n = 3, P < 0.05, Figure 6). Metillikakonitin is a specific blocker of the α7-nicotinic receptor subunit. Adding metillikakonitin at 10 nM increased the amplitude of [Ca²⁺]_i transient by 13 ± 11% (n = 5, P < 0.05, Figure 6), while subsequent addition of nicotine decreased [Ca²⁺]_i transients by 11 ± 5% (n = 6, P < 0.05, Figure 6).

Since both acetylcholine and carbachol reduce [Ca²⁺]_i transient and quantum content of endplate currents, we suggested that cholinergic modulation of neurotransmitter release could result from attenuated Ca²⁺ entry into motor nerve endings (Khaziev et al., 2012). In this work, we found that application of specific N-type Ca²⁺ channel blocker, ω-conotoxin GVIA (300 nM) reduced [Ca²⁺]_i transient by 35 ± 7% (n = 5, P < 0.05, Figures 7A,B). In the presence of ω-conotoxin GVIA carbachol became ineffective (Figures 7C,D), indicating that N-type Ca²⁺-channels play role in presynaptic modulation observed.

Non-specific blocker of all types of nicotinic receptors, d-tubocurarine (10 μM) increased the amplitude of [Ca²⁺]_i transients by 11 ± 3% (n = 15, P < 0.05, Figures 8A,B). Non-specific blocker of muscarinic receptors, atropine (1 μM) also increased [Ca²⁺]_i transient by 9 ± 2% (n = 19, P < 0.05, Figures 8C,D). When applied together, d-tubocurarine and atropine increased the amplitude of [Ca²⁺]_i transients by 7 ± 2% (n = 7, P < 0.05), indicating the absence of additive facilitating effects on [Ca²⁺]_i transient. An increase in Ca²⁺-response in the presence of nicotinic or muscarinic acetylcholine receptor blockers suggests the presence of some tonic concentration of endogenous acetylcholine in the synaptic cleft. This acetylcholine interacts with nicotinic and muscarinic receptors and causes inhibition of Ca²⁺-ions entry into nerve terminal.

It has been shown earlier that anticholinesterase drugs lead to accumulation of endogenous acetylcholine in synaptic cleft (Loewi and Hellauer, 1938; Fatt and Katz, 1952; Fedorov, 1976). At low frequencies of motor nerve stimulation, acetylcholinesterase inhibitor, neostigmine (1 μM), decreased [Ca²⁺]_i transients by 13 ± 5% (n = 5, P < 0.05, Figures 9A,B). This inhibitory effect of neostigmine was fully reversed in the presence of blockers of nicotinic and muscarinic receptors (Figure 9B). We can therefore suggest that endogenous acetylcholine modulates intra-terminal [Ca²⁺]_i transients by acting on presynaptic nicotinic and muscarinic acetylcholine receptors.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.