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.