D. V. Samigullin

Cholinergic regulation of the evoked quantal release at frog neuromuscular junction

The effects of cholinergic drugs on the quantal contents of the nerve‐evoked endplate currents (EPCs) and the parameters of the time course of quantal release (minimal synaptic latency, main modal value of latency histogram and variability of synaptic latencies) were studied at proximal, central and distal regions of the frog neuromuscular synapse. Acetylcholine (ACh, 5 × 10−4m), carbachol (CCh, 1 × 10−5m) or nicotine (5 × 10−6m) increased the numbers of EPCs with long release latencies mainly in the distal region of the endplate (90–120 μm from the last node of Ranvier), where the synchronization of transmitter release was the most pronounced. The parameters of focally recorded motor nerve action potentials were not changed by either ACh or CCh. The effects of CCh and nicotine on quantal dispersion were reduced substantially by 5 × 10−7m (+)tubocurarine (TC). The muscarinic agonists, oxotremorine and the propargyl ester of arecaidine, as well as antagonists such as pirenzepine, AF‐DX 116 and methoctramine, alone or in combination, did not affect the dispersion of the release. Muscarinic antagonists did not block the dispersion action of CCh. Cholinergic drugs either decreased the quantal content mo (muscarinic agonist, oxotremorine M, and nicotinic antagonist, TC), or decreased mo and dispersed the release (ACh, CCh and nicotine). The effects on mo were not related either to the endplate region or to the initial level of release dispersion. It follows that the mechanisms regulating the amount and the time course of transmitter release are different and that, among other factors, they are altered by presynaptic nicotinic receptors.

Modulation of the kinetics of evoked quantal release at mouse neuromuscular junctions by calcium and strontium

The effects of calcium and strontium on the quantal content of nerve‐evoked endplate currents and on the kinetic parameters of quantal release (minimal synaptic delay, value of main mode of synaptic delay histogram, and variability of synaptic delay) were studied at the mouse neuromuscular synapse. At low calcium ion concentrations (0.2–0.6 mmol/L), evoked signals with long synaptic delays (several times longer than the value of main mode) were observed. Their number decreased substantially when [Ca2+]o was increased (i.e. the release of transmitter became more synchronous). By contrast, the early phase of secretion, characterized by minimal synaptic delay and accounting for the main peak of the synaptic delay histogram, did not change significantly with increasing [Ca2+]o. Hence, extracellular calcium affected mainly the late, ‘asynchronous’, portion of phasic release. The average quantal content grew exponentially from 0.09 ± 0.01 to 1.04 ± 0.07 with the increase in [Ca2+]o without reaching saturation. Similar results were obtained when calcium was replaced by strontium, but the asynchronous portion of phasic release was more pronounced and higher strontium concentrations (to 1.2–1.4 mmol/L) were required to abolish responses with long delays. Treatment of preparations with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetrakis acetoxymethyl ester (BAPTA‐AM) (25 µmol/L), but not with ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester (EGTA‐AM) (25 µmol/L), abolished the responses with long delays. The dependence of quantal content and synchrony of quantal release on calcium and strontium concentrations have quite different slopes, suggesting that they are governed by different mechanisms.

Modeling of quantal neurotransmitter release kinetics in the presence of fixed and mobile calcium buffers

The local calcium concentration in the active zone of secretion determines the number and kinetics of neurotransmitter quanta released after the arrival of a nerve action potential in chemical synapses. The small size of mammalian neuromuscular junctions does not allow direct measurement of the correlation between calcium influx, the state of endogenous calcium buffers determining the local concentration of calcium and the time course of quanta exocytosis. In this work, we used computer modeling of quanta release kinetics with various levels of calcium influx and in the presence of endogenous calcium buffers with varying mobilities. The results of this modeling revealed the desynchronization of quanta release under low calcium influx in the presence of an endogenous fixed calcium buffer, with a diffusion coefficient much smaller than that of free Ca2+, and synchronization occurred upon adding a mobile buffer. This corresponds to changes in secretion time course parameters found experimentally (Samigullin et al., Physiol Res 54:129–132, 2005; Bukharaeva et al., J Neurochem 100:939–949, 2007).

Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes

At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers—which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal—has hitherto been technically impossible. With the aim of quantifying both Ca2+ currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 pA and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 μM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

Temperature Effect on Proximal to Distal Gradient of Quantal Release of Acetylcholine at Frog Endplate

The conduction velocity of the nerve terminal, mean quantal content, and release latencies of uniquantal endplate currents (EPCs) were recorded in proximal, central, and distal parts of the terminal by extracellular pipettes located 5, 50, and 100 mm from the end of myelinated nerve trunk. The spike conduction velocity, minimal latency, modal value of the latency histograms, and time interval during which 90% of EPCs released (P90) at distal, central, and proximal part of the frog nerve terminal have different temperature dependency between 10° and 28°C. As shown by the size and time-course of reconstructed multiquantal EPCs, the secretion synchronization, which is greatest in distal parts, compensates at least partly for the progressive slowing of spike conduction velocity in the proximodistal direction, in particular at lower temperatures.

Kinetics of neurotransmitter release in neuromuscular synapses of newborn and adult rats.

The kinetics of the phasic synchronous and delayed asynchronous release of acetylcholine quanta was studied at the neuromuscular junctions of aging rats from infant to mature animals at various frequencies of rhythmic stimulation of the motor nerve. We found that in infants 6 (P6) and 10 (P10) days after birth a strongly asynchronous phase of quantal release was observed, along with a reduced number of quanta compared to the synapses of adults. The rise time and decay of uni‐quantal end‐plate currents were significantly longer in infant synapses. The presynaptic immunostaining revealed that the area of the synapses in infants was significantly (up to six times) smaller than in mature junctions. The intensity of delayed asynchronous release in infants increased with the frequency of stimulation more than in adults. A blockade of the ryanodine receptors, which can contribute to the formation of delayed asynchronous release, had no effect on the kinetics of delayed secretion in the infants unlike synapses of adults. Therefore, high degree of asynchrony of quantal release in infants is not associated with the activity of ryanodine receptors and with the liberation of calcium ions from intracellular calcium stores.

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.

Decreased entry of calcium into motor nerve endings upon activation of presynaptic cholinergic receptors

283 Acetylcholine entering the synaptic cleft from choo linergic nerve endings after depolarization by the action potential not only causes generation of postsynn aptic potential, but also modulates the intensity of the release of subsequent portions of the neurotransmitter, activating presynaptic autoreceptors [1]. Effects of endogenous acetylcholine are reproduced in experii ments, when adding the exogenous acetylcholine or its analogs, including the nonhydrolyzable cholinomii metic carbacholine to the solution perfusing an isoo lated nerve–muscle preparation [2, 3]. Experiments on the frog nerve–muscle preparation show that the addition of carbacholine to the perfusate causes reduction of intensity of evoked secretion of quanta, which is supported by the reduction in the number of quanta (quantum content) released in response to a nerve stimulus, and change the kinetics of release of individual quanta forming multiquantum postsynaptic response. The effect of carbacholine on the quantum content of endplate potentials was decreased in the presence of the nicotinic receptor blocker ddtubb ocurarine [2,3]. Since the processes that are responsii ble both for the quantum content of endplate potential and for the kinetics of quantum release are calciumm dependent (increasing the calcium concentration in the nerve ending increases the quantum content and synchronizes selection of quanta) [4, 5], it is natural to assume that the reduction of entry of calcium in motor nerve ending is the basis of the inhibitory effect of cholinomimetics. To test this hypothesis in the study on isolated nerve–muscle preparations of m. cutaneus pectoris and m. sartorius of the marsh frog Rana ridii bunda, the change in the quantum content of endplate current and the entry of calcium (Ca 2+ transient) in the nerve terminals in response to a single stimulus were compared. The nerve–muscle preparation was continuously perfused with Ringer solution of the following compoo sition content of calcium ions in the medium and the press ence of magnesium prevented muscle contractions in response to nerve stimulation. The nerve was stimuu lated through a suction electrode with 0.22ms rectann gular pulses of a suprathreshold amplitude at a free quency of 0.5 Hz. Recording of miniature and evoked endplate potentials (mEPP and EPP, respectively) was performed by the twooelectrode patch clamp method at a level of –60 mV using an Axoclamp 900A amplii fier and intracellular electrodes filled with a 33M KCl solution with a resistance 5–10 mΩ. The signals were digitized with an ADC LLcard and processed using a software created …

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 β receptors, increased adenylate cyclase activity, and increases in intracellular cAMP levels.

Acetylcholine-Induced Inhibition of Presynaptic Calcium Signals and Transmitter Release in the Frog Neuromuscular Junction

Acetylcholine (ACh), released from axonal terminals of motor neurons in neuromuscular junctions regulates the efficacy of neurotransmission through activation of presynaptic nicotinic and muscarinic autoreceptors. Receptor-mediated presynaptic regulation could reflect either direct action on exocytotic machinery or modulation of Ca2+ entry and resulting intra-terminal Ca2+ dynamics. We have measured free intra-terminal cytosolic Ca2+ ([Ca2+]i) using Oregon-Green 488 microfluorimetry, in parallel with voltage-clamp recordings of spontaneous (mEPC) and evoked (EPC) postsynaptic currents in post-junctional skeletal muscle fiber. Activation of presynaptic muscarinic and nicotinic receptors with exogenous acetylcholine and its non-hydrolized analog carbachol reduced amplitude of the intra-terminal [Ca2+]i transients and decreased quantal content (calculated by dividing the area under EPC curve by the area under mEPC curve). Pharmacological analysis revealed the role of muscarinic receptors of M2 subtype as well as d-tubocurarine-sensitive nicotinic receptor in presynaptic modulation of [Ca2+]i transients. Modulation of synaptic transmission efficacy by ACh receptors was completely eliminated by pharmacological inhibition of N-type Ca2+ channels. We conclude that ACh receptor-mediated reduction of Ca2+ entry into the nerve terminal through N-type Ca2+ channels represents one of possible mechanism of presynaptic modulation in frog neuromuscular junction.