Figure legends
Figure 1. Effects of nicotine on the electrophysiological parameters registered at the mouse neuromuscular junction. Changes in absolute values are shown (a) resting membrane potential (RMP) of muscle fibers, (b) amplitudes of miniature endplate potentials (mEPP), (c) the frequency of the mEPPs and (d) amplitudes of evoked endplate potentials (EPP) in control and 15 min after nicotine application (the range from 0.1 to 50 μM). Results are expressed as mean ± SEM of five independent experiments. Asterisk (*) indicates significant effect (P< 0.05, one-way ANOVA test with Dunnet’s post-hoc comparison).
Figure 2. Nicotine inhibits evoked release of ACh quanta (quantal content, QC) by activating of nNAChRs. Panels on the top are representative traces of EPP and mEPP (50 signals averaged) in separate experiments with nicotine application (Nic, 10 µM; a) and nicotine application after pretreatment with the neuronal cholinergic receptor antagonist DHβE (1 µM; b). (c) Results are expressed as mean ± SEM and SD of QC, as percentage with nicotine (n = 5, 7 NMJs), DHβE (n = 6, 9 NMJs) and DHβE + nicotine (n = 6, 9 NMJs) applications versus control. Asterisk (*) indicates significant effect (P < 0.05, one-way repeated ANOVA test with Tukey’s post-hoc comparison).
Figure 3. Pseudo-color calcium images of a motor nerve terminal loaded with Oregon Green 488 BAPTA-1 Hexapotassium Salt. The axon is imaged before and during single electrical stimulus (0.2 ms duration). Bar is 20 µm.
Figure 4. Nicotine increases the calcium transient in the motor nerve ending by activation of neuronal ACh receptors; blockade of the receptors leads to a decrease in the amplitude of the calcium signal. Panels on the top are representative traces of calcium transient from separate experiments with nicotine application (Nic, 10 µM; a) and nicotine application after pretreatment with nNAChRs antagonist DHβE (1 µM; b). (c) mean ± SEM and SD of the amplitude of the calcium signal, expressed as a percentage of control when applying nicotine (n = 5, 8 NMJs), DHβE (n = 5, 15 NMJs) and DHβE + nicotine (n = 5, 15 NMJs). Asterisk (*) indicates significant effect (P < 0.05, one-way repeated ANOVA test with Tukey’s post-hoc comparison).
Figure 5. The calcium transient-enhancing effect of nicotine is abolished after nonspecific calcium channel blockade, but not after inhibition of Cav2.1 type calcium channels. Panels on the top are representative traces of calcium transient from individual experiments: (a) effect of the nonspecific calcium channel blocker CdCl2 (Cd, 10 µM); (b) no effect of nicotine (Nic, 10 µM) after pretreatment with CdCl2; (c) effect of Cav2.1 VGCCs blocker ω-agatoxin IVA (Aga, 40 nM); and (d) effect of nicotine on the calcium transient after pre-incubation with ω-agatoxin IVA. (e) mean ± SEM and SD of calcium signal amplitudes obtained in the above series and expressed as a percentage of control or value after CdCl2 (n = 5, 9 NMJs), CdCl2+ Nic (n = 5, 9 NMJs), Aga (n = 5, 5 NMJs;) and Aga + Nic (n = 5, 7 NMJs) application. Asterisk (*) indicates significant effect (P< 0.05, one-way repeated ANOVA test).
Figure 6. Lack of the effect of nicotine (an increase in the amplitude of the calcium transient) and DHβE (a decrease in the amplitude of the calcium transient) after blockade of the Cav1 channels. Panels on the top are representative traces of calcium transient from individual experiments: (a) effect of Cav1 calcium channel blocker nitrendipine (Nitre, 25 µM); (b) lack of nicotine (Nic, 10 µM) effect after pre-application of nitrendipine; (c) lack of DHβE (1 µM) effect after nitrendipine pre-treatment; (d) effect of Cav1 type VGCCs blocker verapamil (50 µM); (e) no effect of nicotine after pre-application of verapamil. (f) mean ± SEM and SD of calcium signal amplitudes obtained in the above series and expressed as a percentage of control or value after Nitre (n = 5, 17 NMJs), Nitre + Nic (n = 5, 17 NMJs), Nitre + DHβE (n = 5, 7 NMJs), verapamil (n = 5, 9 NMJs) and verapamil +Nic (n = 5, 9 NMJs) application (five independent experiments). Asterisk (*) indicates significant effect (P< 0.05, one-way repeated ANOVA test).
Figure 7. Nicotine-induced decrease in the ACh release (quantal content, QC) involves L-type Cav1 channels. Panels on the top are representative traces of EPP and mEPP (50 signals averaged) from individual experiments: (a and b) lack of nicotine (Nic, 10 µM) effect after pre-application with Cav1 VGCCs blockers nitrendipine (Nitre, 25 µM) and verapamil (50 µM); (c) mean ± SEM and SD of QC, expressed as a percentage of control or value after Nitre (n = 5, 6 NMJs), Nitre +Nic (n = 5, 6 NMJs), Verapamil (n = 5, 6 NMJs) and Verapamil + Nic (n = 5, 6 NMJs) application (five independent experiments). Asterisk (*) indicates significant effect (P< 0.05, one-way repeated ANOVA test with Tukey’s post-hoc comparison).
Figure 8 - Schematic drawing showing possible mechanisms of the coupling between nAChRs and L-type (Cav1) calcium channels in the motor nerve terminal. (a) Calcium dependent facilitation of the Cav1 type channel, mediated by CaM and triggered by calcium (Kim et al., 2008), which enters through nNAChR; (b) calcium dependent inactivation process of Cav1 type calcium channels, mediated by interaction between CaM and Ca2+channel, is disrupted by an increase in CaMKII activity (Abiria & Colbran, 2010); (c) opening of Cav1 calcium channels and entry of calcium into terminal caused by depolarization due to activation of neuronal AChRs (Katsura et al., 2002).
Figure 9. - Working model of the mechanism of autoregulation of ACh release in the peripheral cholinergic synapse via nNAChRs. Activation of nNAChRs is accompanied by an increase in the entry of calcium ions into the motor nerve terminal through the L-type (Cav1) calcium channels. Latter are involved in both the process of evoked ACh release and its modulation.