ATP Reduces the Entry of Calcium Ions into the Nerve Ending by Blocking L-type Calcium Channels.

At neuromuscular junctions, ATP inhibits both the evoked and spontaneous acetylcholine release and inward calcium current operating via presynaptic P2Y receptors. It was shown in the experiments with the frog neuromuscular synapse using specific calcium-sensitive dye Oregon Green Bapta 1 that exogenous ATP reduces the amplitude of calcium transient, which reflects the changes in the entry of calcium ions in response to the nerve pulse. The depressing effect of ATP on the transient was prevented by suramin, the blocker of P2 receptors. Nitrendipine, a specific blocker of L-type calcium channels, per se decreased the calcium transient amplitude and significantly attenuated the effect of ATP on the calcium signal. Contrariwise, the preliminary application of ATP to the neuromuscular junction completely eliminated the depressing effect of nitrendipine on the calcium response. The obtained data suggest that an essential component in the inhibitory action of ATP on the calcium transient amplitude is provided by reduction of the entry of calcium ions into a frog nerve ending via L-type voltage-gated calcium channels.

Involvement of dihydropyridine-sensitive calcium channels in high asynchrony of transmitter release in neuromuscular synapses of newborn rats.

Experiments on neuromuscular synapses of rats at different stages of ontogenesis have been performed. It has been found that one of the reasons of higher asynchrony of the release of single quanta of acetylcholine in the synapses of newborn animals is the activity of the presynaptic dihydropyridine-sensitive calcium channels of the L-type.

Redox-sensitive synchronizing action of adenosine on transmitter release at the neuromuscular junction.

The kinetics of neurotransmitter release was recognized recently as an important contributor to synaptic efficiency. Since adenosine is the ubiquitous modulator of presynaptic release in peripheral and central synapses, in the current project we studied the action of this purine on the timing of acetylcholine quantal release from motor nerve terminals in the skeletal muscle. Using extracellular recording from frog neuromuscular junction we tested the action of adenosine on the latencies of single quantal events in the pro-oxidant and antioxidant conditions. We found that adenosine, in addition to previously known inhibitory action on release probability, also synchronized release by removing quantal events with long latencies. This action of adenosine on release timing was abolished by oxidants whereas in the presence of the antioxidant the synchronizing action of adenosine was further enhanced. Interestingly, unlike the timing of release, the inhibitory action of adenosine on release probability was redox-independent. Modulation of release timing by adenosine was mediated by purinergic A1 receptors as it was eliminated by the specific A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and mimicked by the specific A1 agonist N(6)-cyclopentyl-adenosine. Consistent with data obtained from dispersion of single quantal events, adenosine also reduced the rise-time of multiquantal synaptic currents. The latter effect was reproduced in the model based on synchronizing effect of adenosine on release timing. Thus, adenosine which is generated at the neuromuscular junction from the breakdown of the co-transmitter ATP induces the synchronization of quantal events. The effect of adenosine on release timing should preserve the fidelity of synaptic transmission via "cost-effective" use of less transmitter quanta. Our findings also revealed important crosstalk between purinergic and redox modulation of synaptic processes which could take place in the elderly or in neuromuscular diseases associated with oxidative stress like lateral amyotrophic sclerosis.

Voltage-dependent P/Q-type calcium channels at the frog neuromuscular junction.

It is well known that antagonists of N-type voltage-gated calcium channels inhibit the evoked quantal release of acetylcholine in amphibian neuromuscular synapses. This, however, does not exclude the functional expression of other types of voltage-gated calcium channels in these nerve terminals. Using immunocytochemistry, we detected the expression of the alpha1A subunit of P/Q-type calcium channels (that is otherwise typical of mammalian motor nerve endings) in the frog neuromuscular junction. In addition, we demonstrated that the P/Q-type channel blocker omega-agatoxin IVA (20 nM) reduced the action potential-induced calcium transient and significantly decreased both spontaneous and evoked mediator release. Our data indicates the functional expression of P/Q-type calcium channels in the frog motor nerve ending which participate in acetylcholine release.

Opposite modulation of time course of quantal release in two parts of the same synapse by reactive oxygen species.

Reactive oxygen species (ROS) are potent regulators of transmitter release in chemical synapses, but the mechanism of this action remains almost unknown. Presynaptic modulation can change either the release probability or the time course of quantal release, which was recently recognized as an efficient mechanism determining synaptic efficiency. The nonuniform structure and a big size of the frog neuromuscular junction make it a useful model to study the action of ROS in compartments different in release probability and in time course of transmitter release. The time course (or kinetics) of quantal release could be estimated by measuring the dispersion of the synaptic delays for evoked uniquantal endplate currents (EPCs) under low release probability. Using two-electrode recording technique, the action of ROS on kinetics and release probabilities were studied at the proximal and distal parts within the same neuromuscular junction. The stable ROS hydrogen peroxide (H2O2) increased the dispersion of synaptic delays of EPCs (i.e. desynchronized quantal release) within the distal part but decreased delay dispersion (synchronized quantal release) within the proximal part of the same synapse. Unlike the opposite modulation of kinetics, H2O2 reduced release probability in both distal and proximal parts. Since ATP is released from motor nerve terminals together with acetylcholine and can be involved in ROS signaling, we tested the presynaptic action of ATP. In the presence of the pro-oxidant Fe2+, extracellular ATP, similarly to H2O2, induced significant desynchronization of release in the distal regions. The antioxidant N-acetyl-cysteine attenuated the inhibitory action of ATP on release probability and abolished the action of H2O2 and ATP in the presence of Fe2+, on release kinetics. Our data suggest that ROS induced during muscle activity could change the time course of transmitter release along the motor nerve terminal to provide fine tuning of synaptic efficacy.

[Calcium modulation of the release kinetics of the acetylcholine quanta generating multiquantal postsynaptic response].

The effects of calcium on the quantal content of nerve-evoked endplate currents (EPC) and on the temporal parameters of quantal release were studied in the frog neuromuscular synapse using the method of "subtractions". It was shown that under physiological conditions quanta generating multiquantal postsynaptic responses were released nonsynchronously because of a considerable variability of latencies of the uniquantal responses forming multiquantal EPC. Different calcium dependences for EPCs quantal content and time course of the quantal release were revealed. The average quantal content grew exponentially with the increase in calcium concentration from 0.4 to 1.8 mmol/L, whereas the release synchronicity reached the maximum at 1 mmol/L calcium. It was suggested that the changes in the synchronicity of the evoked release were one of the mechanisms of the synaptic plasticity.

[Changes in the kinetics of quanta secretion-effective mechanism of synaptic transmission modulation].

It is widely accepted that the leading presynaptic mechanisms underlying the synaptic plasticity involve changes of the number of neurotransmitter quanta released by one nerve pulse (the quantal content of postsynaptic response) and of the size of a single quantum. In addition, the existence of one more effective though previously ignored mechanism of modulation of synaptic plasticity was suggested related to the change in the time course (kinetics) of secretion of single neurotransmitter quanta forming the multiquantal response. This article reviews current data (including the authors' own results) on the kinetics of evoked neurotransmitter quanta secretion from motor nerve endings in peripheral synapses, mechanisms of their modulation and methods of quantitative analysis.

Different sensitivity of miniature endplate currents in rat external and internal intercostal muscles to the acetylcholinesterase inhibitor C-547 as compared with diaphragm and extensor digitorum longus.

Derivative of 6-methyluracil, selective cholinesterase inhibitor C-547 potentiates miniature endplate currents (MEPCs) in rat external intercostal muscles (external ICM) more effectively than in internal intercostal muscles (internal ICM). Effect of the C-547 on intercostal muscles was compared with those on extensor digitorum longus (EDL) and diaphragm muscles. Half-effective concentrations for tau of MEPC decay arranged in increasing order were as follows: EDL, locomotor muscle, most sensitive = 1.3 nM, external ICM, inspiration muscle = 6.8 nM, diaphragm, main inspiration muscle = 28 nM, internal ICM, expiration muscle = 71 nM. External ICM might therefore be inhibited, similarly as the limb muscles, by nanomolar concentrations of the drug and do not participate in inspiration in the presence of the C-547. Moreover, internal ICM inhibition can hinder the expiration during exercise-induced fast breathing of C-547- treated experimental animals.

Recovery of mouse neuromuscular junctions from single and repeated injections of botulinum neurotoxin A.

Botulinum neurotoxin type A (BoNT/A) paralyses muscles by blocking acetylcholine (ACh) release from motor nerve terminals. Although highly toxic, it is used clinically to weaken muscles whose contraction is undesirable, as in dystonias. The effects of an injection of BoNT/A wear off after 3-4 months so repeated injections are often used. Recovery of neuromuscular transmission is accompanied by the formation of motor axon sprouts, some of which form new synaptic contacts. However, the functional importance of these new contacts is unknown. Using intracellular and focal extracellular recording we show that in the mouse epitrochleoanconeus (ETA), quantal release from the region of the original neuromuscular junction (NMJ) can be detected as soon as from new synaptic contacts, and generally accounts for > 80% of total release. During recovery the synaptic delay and the rise and decay times of endplate potentials (EPPs) become prolonged approximately 3-fold, but return to normal after 2-3 months. When studied after 3-4 months, the response to repetitive stimulation at frequencies up to 100 Hz is normal. When two or three injections of BoNT/A are given at intervals of 3-4 months, quantal release returns to normal values more slowly than after a single injection (11 and 15 weeks to reach 50% of control values versus 6 weeks after a single injection). In addition, branching of the intramuscular muscular motor axons, the distribution of the NMJs and the structure of many individual NMJs remain abnormal. These findings highlight the plasticity of the mammalian NMJ but also suggest important limits to it.

[The influence of hindlimb unloading on the effectiveness of modulation of the mediator secretion via the autoreceptor system].

In experiments on neuromuscular synapses of rat fast (m. Extensor digitorum longus, EDL) and slow (m. soleus) skeletal muscles, changes in the intensity of spontaneous quantal mediator secretion in response to the activation of presynaptic cholinoreceptors by the nonhydrolyzable acetylcholine analogue carbachol and to an increase in K+ concentration in the control group of animals and in animals subjected to different terms of unloading of hindlimbs have been compared. The intensity of spontaneous secretion of mediator quanta was evaluated from the mean frequency of miniature endplate potentials. In the control group of animals, the frequency of miniature endplate potentials by the action of carbachol increased by 363% in m. EDL and by 62% in m. soleus. The frequency of miniature endplate potentials in the synapses of m. EDL was more sensitive to K(+)-induced depolarization too. The bearing unloading of hindlimbs abolished the sensitivity of spontaneous secretion to carbachol in the synapses of m. EDL, whereas in m. soleus it was unchanged. However, the preservation of sensitivity of nerve endings of fast muscle to K(+)-induced depolarization allows one to assume that the hindlimb unloading leads to a decrease in the number of functioning presynaptic receptors.

[Mechanism of carbachol and adenosine action on spontaneous quantal mediator release at frog neuromuscular junction].

Experiments on the frog sartorius muscle showed that nonhydrolisable acetylcholine analog carbachol (CCh) depresses spontaneous quantal mediator release via muscarinic M2 receptors of nerve ending. Adenosine (Ade) acting via inhibitory A1 receptors is another strong spontaneous quantal release modulator. Inhibition of pertussis toxin (PTx)-sensitive G-proteins only partly eliminated CCh and Ade depressive action. It means metabotropic A1 and M2 receptors of the frog nerve ending regulate spontaneous quantal release via activating of both PTx-sensitive and PTx-insensitive inhibitory mechanisms.

Negative cross-talk between presynaptic adenosine and acetylcholine receptors.

Functional interactions between presynaptic adenosine and acetylcholine (ACh) autoreceptors were studied at the frog neuromuscular junction by recording miniature end-plate potentials (MEPPs) during bath or local application of agonists. The frequency of MEPPs was reduced by adenosine acting on presynaptic adenosine A1 receptors (EC(50) = 1.1 microm) or by carbachol acting on muscarinic M2 receptors (EC(50) = 1.8 microm). However, carbachol did not produce the depressant effect when it was applied after the action of adenosine had reached its maximum. This phenomenon implied that the negative cross-talk (occlusion) had occurred between A1 and M2 receptors. Moreover, the occlusion was receptor-specific as ATP applied in the presence of adenosine continued to depress MEPP frequency. Muscarinic antagonists [atropine or 1-[[2-[(diethylamino)methyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepine-6-one) (AFDX-116)] had no effect on the inhibitory action of adenosine and adenosine antagonists [8-(p-sulfophenyl)theophylline (8-SPT) or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX)] had no effect on the action of carbachol. These data suggested that membrane-delimited interactions did not occur between A1 and M2 receptors. Both carbachol and adenosine similarly inhibited quantal release triggered by high potassium, ionomycin or sucrose. These results indicated a convergence of intracellular pathways activated by M2 and A1 receptors to a common presynaptic effector located downstream of Ca(2+) influx. We propose that the negative cross-talk between two major autoreceptors could take place during intense synaptic activity and thereby attenuate the presynaptic inhibitory effects of ACh and adenosine.

Different sensitivity of miniature endplate currents of the rat extensor digitorum longus, soleus and diaphragm muscles to a novel acetylcholinesterase inhibitor C-547.

A novel derivative of 6-methyluracil, C-547, increased the amplitude and prolonged the duration of miniature endplate currents (MEPCs) which is typical for acetylcholinesterase inhibition. In the soleus and extensor digitorum longus significant potentiation was detected at nanomolar concentrations. In contrast, in the diaphragm muscle, the increase in the amplitudes of the MEPCs and the decay time constant appeared only when the concentration of C-547 was elevated to 1 x 10(-7) M. Possible consequences for the exploitation of this drug, which can selectively inhibit AChE in particular synapses, are discussed.

Spontaneous quantal and non-quantal release of acetylcholine at mouse endplate during onset of hypoxia.

At 20 (0)C, both quantal and non-quantal spontaneous acetylcholine release (expressed as miniature endplate potential frequency [f-MEPPs] and the H-effect, respectively) increased during the first 30 min of hypoxia in solution with normal extracellular calcium ([Ca(2+)](o) = 2.0 mM). The hypoxia-induced tenfold increase of the f-MEPPs was virtually absent in low calcium solution([Ca(2+)](o) = 0.4 mM) whereas there was still a significant increment of non-quantal release. This indicates that each of these two processes of acetylcholine release is influenced by mechanisms with different oxygen sensitivity. The rise of f-MEPPs during the onset of hypoxia apparently requires Ca(2+) entry into the nerve terminal, whereas the non-quantal release can be increased by another factors such as a lower level of ATP.

Calcium dependence of uni-quantal release latencies and quantal content at mouse neuromuscular junction.

Uni-quantal endplate currents (EPC) were recorded at mouse diaphragm neuromuscular synapse by extracellular microelectrode during motor nerve stimulation. The probability of release expressed as quantal content m(o), and variability of synaptic latencies expressed as P90 were estimated in the presence of extracellular calcium ([Ca2+]o) varying between 0.2 and 0.6 mM in the bathing solution. At 0.2 mM ([Ca2+]o), m(o) was low (0.10) and many of long-latency EPCs were present during the late phase of the release (P90 = 2.44 ms). No change in m(o) was found when ([Ca2+]o) was 0.3 mM, but P90 decreased by 39 %. For latency shortening, saturating concentration of ([Ca2+]o) was 0.4 mM, when P90 was 1.49 ms and latencies did not further change at 0.5 and 0.6 mM ([Ca2+]o). In the latter concentrations, however, an increase of m(o) was still observed. It can be concluded that the early phase of the secretion did not significantly change when ([Ca2+]o) was raised and that only the late phase of the release depends on extracellular calcium up to 0.4 mM.