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.

Synaptosome-associated protein 25 (SNAP25) synthesis in terminal buttons of mouse motor neuron.

Previously, we formulated the hypothesis of compartmentalized protein synthesis in axons of motor neurons. In the axon hillock, along the entire length of the axon and in its ending, specific proteins are locally synthesized, which ensure the function of each compartment. In support of this hypothesis, in this work we studied the local protein synthesis in mouse motor nerve ending.

[Neuromuscular synaptic transmission at different stages of postnatal development in rats].

On the nerve-muscle preparation of rats diaphragm muscle on different stages of postnatal development, the comparison of morphological features and functions of synaptic apparatus, including induced secretion time parameters was carried out. It was found that, along with the reduced, compared to the adult animals, area of nerve endings in the newborn the speed of the motor nerve excitation was slower, intensity of spontaneous and induced secretion of quantum fluctuations was reduced and real synaptic delays in the end plate were intense. Severe degree of acetylcholine quanta asynchronous secretion with longer open state of the ion channel in newborns synapses can compensate reduction in reliability of synaptic transmission due to a decrease of the quantal content of the postsynaptic response.

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.

The effects of carbachol on the proximal and distal parts of frog motor nerve endings.

Cholinomimetics not only activate postsynaptic cholinoreceptors in neuromuscular synapses, but also alter the process of acetylcholine secretion from nerve endings. However, the mechanism of action of cholinomimetics on the secretory process remains unidentified. We approached the question of the mechanism of the presynaptic action of cholinomimetics in the present study by investigating the effects of the n,m-cholinomimetic carbachol on nerve ending currents and postsynaptic membrane currents. Carbachol induced decreases in the postsynaptic response, without affecting the duration and amplitude of the nerve ending current in both the central and distal part of the nerve ending. However, carbachol increased the time between the arrival of the presynaptic action potential and the start of transmitter secretion. This effect on synaptic delay was more marked in the distal parts of the ending. The action of another potential modulator, extracellular potassium, was accompanied by decreases in presynaptic currents and also by increases in synaptic delay. These data provide evidence for the suppressive effect of carbachol on acetylcholine secretion acting via presynaptic metabotropic cholinoreceptors which control the level and time course of secretion of neurotransmitter quanta.

Characteristics of the time course of evoked secretion of transmitter quanta in different parts of the motor nerve ending in the frog.

Experiments were performed on neuromuscular preparations from frogs, in which three extracellular microelectrodes were used to record nerve ending currents and single-quantum endplate currents simultaneously from the proximal, central, and distal parts of single synaptic contacts. The rate of propagation of excitation across terminals was measured. along with the minimum synaptic delay, the intensity. and the degree of synchronicity of the secretion of transmitter quanta in different parts of the nerve ending, and the relationships between these factors and the calcium ion concentration in the medium. These studies showed that along with gradients in the rate of conduction of excitation and the intensity of secretion in different parts of the ending. there were also differences in the kinetics of the release of transmitter quanta. As the distance from the end of the myelinated part of the axon increased, the rate of conduction of the nerve impulse and the duration of the synaptic delay decreased, while the synchronicity of the release of quanta increased. Increases in the calcium concentration in the medium produced greater increases in the synchronicity of transmitter quantum release in the distal parts of the synapse than in the proximal parts. Mathematical modeling of multiple-quantum endplate currents showed that the characteristics of the kinetics of the secretion process observed here in different parts of the nerve ending represent a factor which partially compensates for the decrease in the amplitude and extending of the duration of the leading front of the multiple-quantum endplate current which are associated with the low rate of conduction of excitation across the nerve ending. The contribution of this compensation increases as the intensity of secretion of transmitter quanta increases in the distal parts of the synaptic contact.

The role of intracellular cAMP in mediating the synchronizing action of noradrenaline on the evoked release of quanta of mediator in the frog synapse.

Experiments on frog neuromuscular junction preparations with extracellular recording of action currents in nerve endings and single-quantum currents from endplates were used to assess the time course of evoked quantum mediator secretion by analyzing histograms showing the distribution of true synaptic delays. Studies using the cyclic AMP analog dibutyryl-cAMP (db-cAMP), the adenylate cyclase activator forskolin, and the nucleotide-dependent phosphodiesterase inhibitor isobutylmethylxanthine, showed that these agents, like noradrenaline, altered the kinetics of secretion of quanta, leading to synchronization of the release of mediator. After preliminary treatment of the neuromuscular preparation with db-cAMP, forskolin, or isobutylmethylxanthine, noradrenaline did not induce the synchronization of mediator release in quanta. It was concluded that the action of noradrenaline on the time course of secretion is mediated by activation of presynaptic beta receptors, increased adenylate cyclase activity, and increases in intracellular cAMP levels.

[Effect of carbacholine on proximal and distal regions of motor nerve terminals in the frog]. / Vliianie karbakholina na proksimal'nye i distal'nye uchastki dvigatel'nykh nervnykh okonchanii liagushki.

Carbacholine depressed postsynaptic currents in the frog m. sartorius leaving intact presynaptic currents in proximal and distal portions of the motor nerve ending. The carbacholine depressing action was followed by an increase in the time gap between the beginning of presynaptic depolarisation and subsequent quantal release. This effect was considerably more obvious in the distal portions of the nerve endings. Effect of extracellular potassium was evident in a diminishing of presynaptic currents due to membrane depolarisation. The data obtained suggest that carbacholine presynaptically depresses synaptic transmission via metabotropic cholinergic receptors controlling the time course of the transmitter release.

[The time course of the evoked secretion of the mediator quanta in various regions of the frog motor nerve ending]. / Osobennosti vremennogo khoda vyzvannoi sekretsii kvantov mediatora v raznykh otdelakh dvigatel'nogo nervnogo okonchaniia liagushki.

Apart from the fact that the gradient of the velocity of the AP propagation along the nerve terminal and the intensity of secretion do exist, the kinetics of a quanta transmitter release may also be revealed in different parts of the terminal. The velocity of the propagation and the minimum sympatric delay tend to diminish along with moving away from the myelinated part of axon, whereas the synchronicity of the quanta release rises. The distinctions in the time course of secretion in different parts of the terminal were amplified when the calcium ion concentration in the medium was enhanced. The observed peculiarities of the secretion kinetics in different regions of nerve ending seem to compensate for diminishing of the amplitude of multiquantal endplate current.

[Intracellular cAMP involvement in the synchronized activity of noradrenaline in response to evoked release of the transmitter quanta in the frog synapses]. / Uchastie vnutrikletochnogo tsAMF v realizatsii sinkhroniziruiushchego deistviia noradrenalina na vyzvannoe osvobozhdeniie kvantov mediatora v sinapsakh liagushki.

An analogue of cyclic AMP (db-cAMP) penetrating into the frog neuromuscular junction's cell, as well as the adenylyl cyclase activator forskolin, and inhibitor of nucleotide-depending phosphodiesterase isobutilmethylxantine alter the kinetics of the quanta secretion resulting in synchronizing of the process of the transmitter release. Following a db-cAMP preliminary action, no such synchronizing of the transmitter release occurred. Action of noradrenaline on the time course of the secretion seems to be realised through activation of presynaptic beta-adrenoreceptors, augmentation of the adenylyl cyclase activity, and the rise of the intracellular cAMP.