Eugeny E. Nikolsky

Development of desensitization during repetitive end‐plate activity and single end‐plate currents in frog muscle.

1. The amplitudes of end‐plate currents (EPCs) in short trains of fifteen to seventeen EPCs at 10 Hz were depressed in the presence of 10 microM‐proadifen when acetylcholinesterase (AChE) was inhibited. 2. The proadifen‐induced EPC depression was voltage‐dependent and the effect was more pronounced at negative membrane potentials. 3. In the presence of proadifen, the mean amplitude of miniature end‐plate currents (MEPCs) was reduced by 36% 5 s after the EPC train as compared with MEPCs before the train. 4. Without proadifen, but with inhibited AChE, an increase of temperature from 20 to 26 degrees C and elevation of external Ca2+ from 1.8 to 2.5 mM led to EPC amplitude depression in the train, which was also potential‐dependent. 5. After AChE inhibition, proadifen (10 microM) progressively shortened MEPC decay without significant reduction of amplitude up to 40 min of exposition. MEPCs were not affected by proadifen when AChE was active. 6. It is concluded that these postsynaptic effects of proadifen can be explained neither by its action on the resting acetylcholine receptors (AChR) nor on open ion channels but are due to its desensitization‐promoting action.

Role of non‐quantal acetylcholine release in surplus polarization of mouse diaphragm fibres at the endplate zone.

1. In mouse diaphragm, with intact cholinesterase (ChE), the mean value of the resting membrane potential was significantly higher (‐84.8 +/‐ 0.3 mV; mean +/‐ S.E.M.) at the endplate zone than in the extrajunctional area of the muscle fibres (‐82.5 +/‐ 0.3 mV) at 22 degrees C. 2. This hyperpolarization of about 2‐3 mV at the endplate zone was abolished within 5 min by 1 x 10(‐6) M ouabain, indicating that it might be caused by an electrogenic Na(+)‐K+ pump. (+)‐Tubocurarine (TC; 1 x 10(‐5) M) had no effect on this hyperpolarization after bath application for 10‐20 min. 3. Short‐term denervation (4 h), a slight increase of Mg2+ in the bath of from 1 to 4 mM and application of a Ca(2+)‐free solution for 60 min also led to the disappearance of the surplus polarization. All of these factors are known to eliminate TC‐induced hyperpolarization in anti‐ChE‐treated muscles (H‐effect), which is considered to be a correlate of non‐quantal acetylcholine (ACh) leakage. 4. The time courses of the decline of the H‐effect and surplus polarization after denervation were identical. 5. In short‐term denervated muscles with intact ChE, the surplus polarization was restored by 5 x 10(‐8) M ACh, which simulates the H‐effect in anti‐ChE‐treated muscles. The presence of 1 x 10(‐6) M ouabain either prevented or abolished the effect of the bath‐applied ACh.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase A cascade regulates quantal release dispersion at frog muscle endplate

Uniquantal endplate currents (EPCs) were recorded simultaneously at the proximal, central and distal parts of the frog neuromuscular synapse, and their minimal synaptic latencies, latency dispersions and sensitivity to noradrenaline, cAMP and protein kinase A inhibition were measured. The latency dispersion was highest in the proximal part (P90= 1.25 ms); it decreased to P90= 0.95 ms in the central part and to P90= 0.75 ms (60 % of the proximal part) in the distal part. In the proximal parts of the long neuromuscular synapse, stimulation‐evoked EPCs with long release latencies were eliminated when the intracellular cAMP was increased by β1 activation by noradrenaline, by the permeable analogue db‐cAMP, by activation of adenylyl cyclase or by inhibition of cAMP hydrolysis. This makes the evoked release more compact, and the amplitude of the reconstructed multiquantal currents increases. Protein kinase A is a target of this regulation, since a specific inhibitor, Rp‐cAMP, prevents the action of cAMP in the proximal parts and increases the occurrence of long‐latency events in the distal parts of the synapse. Our results show that protein kinase A is involved in the timing of quantal release and can be regulated by presynaptic adrenergic receptors.

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.

The effect of non-quantal acetylcholine release on quantal miniature currents at mouse diaphragm.

1. The amplitude and exponential decay time constant of miniature endplate currents (MEPCs) were measured in mouse diaphragms treated with anti‐cholinesterase under conditions known to modulate non‐quantal acetylcholine (ACh) release. 2. Anti‐cholinesterase prolonged MEPC decay and the extent of this initial prolongation was not influenced by non‐quantal release. When non‐quantal release was present, the decays of MEPCs became increasingly faster over several hours. This increased decay did not occur in the absence of non‐quantal release. 3. Potentiation of the non‐quantal release by zero Mg2+ and 1 x 10(‐5) M choline, on the other hand, led to acceleration of MEPC shortening. 4. Increase of temperature from 15 to 26 degrees C and the presence of the desensitization‐promoting drug proadifen (5 x 10(‐6) M) accelerated the rate of MEPC shortening. 5. These observations are consistent with increased receptor desensitization due to non‐quantal release. Repetitive binding of ACh to postsynaptic receptors which prolongs the time course of MEPC in anti‐cholinesterase‐treated endplates leads to progressive desensitization in the presence of non‐quantal release and to the subsequent shortening of the quantal responses.

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).

Purine P2Y receptors in ATP-mediated regulation of non-quantal acetylcholine release from motor nerve endings of rat diaphragm.

We established the effect of ATP, which is released together with acetylcholine (ACh), on the non-quantal ACh release (NQR) in rat diaphragm endplates and checked what kind of purine receptors are involved. NQR was estimated by the amplitude of endplate hyperpolarization (the H-effect) following the blockade of postsynaptic nicotinic receptors and cholinesterase. 100 μM ATP reduced the H-effect to 66% of the control. The action of ATP remained unchanged after the inhibition of ionotropic P2X receptors by Evans blue and PPADS, but disappeared after the application of the broad spectrum P2 receptor antagonist suramin, metabotropic P2Y receptor blocker reactive blue 2 and U73122, an inhibitor of phospholipase C. P2Y-mediated regulation is not coupled to presynaptic voltage-dependent Ca(2+) channels. During the simultaneous application of ATP and glutamate (which is another ACh cotransmitter reducing non-quantal release), the additive depressant effect led to a disappearance of the H-effect. This can be explained by the independence of the action of ATP and glutamate. Unlike the effects of purines on the spontaneous quantal secretion of ACh, its non-quantal release is regulated via P2Y receptors coupled to G(q/11) and PLC. ATP thus regulates the neuromuscular synapse by two different pathways.

Different sensitivities of rat skeletal muscles and brain to novel anti‐cholinesterase agents, alkylammonium derivatives of 6‐methyluracil (ADEMS)

The rat respiratory muscle diaphragm has markedly lower sensitivity than the locomotor muscle extensor digitorum longus (EDL) to the new acetylcholinesterase (AChE) inhibitors, alkylammonium derivatives of 6‐methyluracil (ADEMS). This study evaluated several possible reasons for differing sensitivity between the diaphragm and limb muscles and between the muscles and the brain.

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.