Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction.

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Muscarinic cholinoreceptors (M1-, M2-, M3- and M4-type) modulate the acetylcholine secretion in the frog neuromuscular junction.

Muscarinic cholinoreceptors regulate the neurosecretion process in vertebrate neuromuscular junctions. The diversity of muscarinic effects on acetylcholine (ACh) secretion may be attributed to the different muscarinic subtypes involved in this process. In the present study, the location of five muscarinic receptor subtypes (M1, M2, M3, M4 and M5) on the motor nerve terminals of frog cutaneous pectoris muscle was shown using specific polyclonal antibodies. The modulatory roles of these receptors were investigated via assessment of the effects of muscarine and specific muscarinic antagonists on the quantal content of endplate currents (EPCs) and the time course of secretion, which was estimated from the distribution of "real" synaptic delays of EPCs recorded in a low Ca2+/high Mg2+ solution. The agonist muscarine decreased the EPC quantal content and synchronized the release process. The depressing action of muscarine on the EPC quantal content was abolished only by pretreatment of the preparation with the M3 blockers 4-DAMP (1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide) and J 104129 fumarate ((αR)-α-Cyclopentyl-α-hydroxy-N-[1-(4-methyl-3-pentenyl)-4-piperidinyl]benzeneacetamide fumarate). Moreover, antagonists of the M1, M2, M3 and M4 receptors per se diminished the intensity of secretion, which suggests a putative up-regulation of the release by endogenous ACh.

Age-dependent action of reactive oxygen species on transmitter release in mammalian neuromuscular junctions.

Reactive oxygen species (ROS) are implicated in aging, but the neurobiological mechanisms of ROS action are not fully understood. Using electrophysiological techniques and biochemical assays, we studied the age-dependent effect of hydrogen peroxide (H2O2) on acetylcholine release in rat diaphragm neuromuscular junctions. H2O2 significantly inhibited both spontaneous (measured as frequency of miniature end-plate potentials) and evoked (amplitude of end-plate potentials) transmitter release in adult rats. The inhibitory effect of H2O2 was much stronger in old rats, whereas in newborns tested during the first postnatal week, H2O2 did not affect spontaneous release from nerve endings and potentiated end-plate potentials. Proteinkinase C activation or intracellular Ca2+ elevation restored redox sensitivity of miniature end-plate potentials in newborns. The resistance of neonates to H2O2 inhibition was associated with higher catalase and glutathione peroxidase activities in skeletal muscle. In contrast, the activities of these enzymes were downregulated in old rats. Our data indicate that the vulnerability of transmitter release to oxidative damage strongly correlates with aging and might be used as an early indicator of senescence.

Bayesian analysis of the kinetics of quantal transmitter secretion at the neuromuscular junction.

The timing of transmitter release from nerve endings is considered nowadays as one of the factors determining the plasticity and efficacy of synaptic transmission. In the neuromuscular junction, the moments of release of individual acetylcholine quanta are related to the synaptic delays of uniquantal endplate currents recorded under conditions of lowered extracellular calcium. Using Bayesian modelling, we performed a statistical analysis of synaptic delays in mouse neuromuscular junction with different patterns of rhythmic nerve stimulation and when the entry of calcium ions into the nerve terminal was modified. We have obtained a statistical model of the release timing which is represented as the summation of two independent statistical distributions. The first of these is the exponentially modified Gaussian distribution. The mixture of normal and exponential components in this distribution can be interpreted as a two-stage mechanism of early and late periods of phasic synchronous secretion. The parameters of this distribution depend on both the stimulation frequency of the motor nerve and the calcium ions' entry conditions. The second distribution was modelled as quasi-uniform, with parameters independent of nerve stimulation frequency and calcium entry. Two different probability density functions for the distribution of synaptic delays suggest at least two independent processes controlling the time course of secretion, one of them potentially involving two stages. The relative contribution of these processes to the total number of mediator quanta released depends differently on the motor nerve stimulation pattern and on calcium ion entry into nerve endings.

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 Ca(2+) 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.

Effect of tissue-specific acetylcholinesterase inhibitor C-547 on α3ß4 and αßεδ acetylcholine receptors in COS cells.

The C-547 is the most effective muscle and tissue-specific anticholinesterase among alkylammonium derivatives of 6-methyluracil (ADEMS) acting in nanomolar concentrations on locomotor muscles but not on respiratory muscles, smooth muscles and heart and brain acetylcholine esterases (AChE). When applied systematically it could influence peripheral acetylcholine receptors. The aim of the present study was to investigate the effect of C-547 on rat α3ß4 (ganglionic type) and αßεδ (muscle type) nicotinic receptors expressed in COS cells. Currents evoked by rapid application of acetylcholine or nicotine were recorded in whole-cell mode by electrophysiological patch-clamp technique 2-4 days after cell transfection by plasmids coding the α3ß4 or αßεδ combination of receptor subunits. In cells sensitive to acetylcholine, the application of C-547 evoked no responses. When acetylcholine was applied during an already running application of C-547, acetylcholine responses were only inhibited at concentrations higher than 10(-7)M. This inhibition is not voltage-dependent, but is accompanied by an increased rate of desensitization. Thus in both types of receptors, effective doses are approximately 100 times higher than those inhibiting AChE in leg muscles and similar to those inhibiting respiratory diaphragm muscles and external intercostal muscles. These observations show that C-547 can be considered for symptomatic treatment of myasthenia gravis and other congenital myasthenic syndromes as an inhibitor of AChE in leg muscles at concentrations much lower than those inhibiting muscle and ganglion types of acetylcholine receptors.

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).

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: Increased amplitude and prolonged decay time of miniature endplate currents were used to assess anti-cholinesterase activity in muscles. In hippocampal slices, induction of synchronous network activity was used to follow cholinesterase inhibition. The inhibitor sensitivities of purified AChE from the EDL and brain were also estimated. KEY RESULTS: The intermuscular difference in sensitivity to ADEMS is partly explained caused by a higher level of mRNA and activity of 1,3-bis[5(diethyl-o-nitrobenzylammonium)pentyl]-6-methyluracildibromide (C-547)-resistant BuChE in the diaphragm. Moreover, diaphragm AChE was more than 20 times less sensitive to C-547 than that from the EDL. Sensitivity of the EDL to C-547 dramatically decreased after treadmill exercises that increased the amount of PRiMA AChE(G4), but not ColQ AChE(A12) molecular forms. The A12 form present in muscles appeared more sensitive to C-547. The main form of AChE in brain, PRiMA AChE(G4), was apparently less sensitive because brain cholinesterase activity was almost three orders of magnitude more resistant to C-547 than that of the EDL. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that ADEMS compounds could be used for the selective inhibition of AChEs and as potential therapeutic tools.

Mechanisms of carbacholine and GABA action on resting membrane potential and Na+/K+-ATPase of Lumbricus terrestris body wall muscles.

This work was aimed to identify the action of several ion channel and pump inhibitors as well as nicotinic, GABAergic, purinergic and serotoninergic drugs on the resting membrane potential (RMP) and assess the role of cholinergic and GABAergic sensitivity in earthworm muscle electrogenesis. The nicotinic agonists acetylcholine (ACh), carbacholine (CCh) and nicotine depolarize the RMP at concentrations of 5 µM and higher. The nicotinic antagonists (+)tubocurarine, α-bungarotoxin, muscarinic antagonists atropine and hexamethonium do not remove or prevent the CCh-induced depolarization. Verapamil, tetrodotoxin, removal of Cl(-) and Ca(2+) from the solution also cannot prevent the depolarization by CCh. In a Na(+)-free medium, however, CCh lost this depolarization ability and this indicates that the drug opens the sodium permeable pathway. Serotonin, glutamate, glycine, adenosine triphosphate (ATP) and cis-4-aminocrotonic acid (GABA(C) receptor antagonist) had no effect on the RMP. On the other hand, isoguvacin, γ-aminobutyric acid (GABA) and baclofen (GABA(B) receptor agonist) hyperpolarized the RMP. Ouabain, bicucullin (GABA(A) antagonist) and phaclofen (GABA(B) antagonist), as well as the removal of Cl(-), suppressed the effect of GABA and baclofen. CCh did not enhance the depolarization generated by ouabain but, on the other hand, hindered the hyperpolarizing activity of baclofen both in the absence and presence of atropine and (+)tubocurarine. The long-term application of CCh depolarizes the RMP primarily by inhibiting the Na(+)/K(+)-ATPase. The muscle membrane also contains A and B type GABA binding sites, the activation of which increases the RMP at the expense of increasing the action of ouabain- and Cl(-) -sensitive electrogenic pumps.

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 Ca(2+), 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).

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 [Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](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 micromol/L), but not with ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM) (25 micromol/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.

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 x 10(-4) M), carbachol (CCh, 1 x 10(-5) M) or nicotine (5 x 10(-6) M) increased the numbers of EPCs with long release latencies mainly in the distal region of the endplate (90-120 microm 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 x 10(-7) M (+)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 m(o) (muscarinic agonist, oxotremorine M, and nicotinic antagonist, TC), or decreased m(o) and dispersed the release (ACh, CCh and nicotine). The effects on m(o) 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.

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 beta1 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.