The Mechanism of α2 adrenoreceptor-dependent Modulation of Neurotransmitter Release at the Neuromuscular Junctions.

α2-Adrenoreceptors (ARs) are main Gi-protein coupled autoreceptors in sympathetic nerve terminals and targets for dexmedetomidine (DEX), a widely used sedative. We hypothesize that α2-ARs are also potent regulators of neuromuscular transmission via G protein-gated inwardly rectifying potassium (GIRK) channels. Using extracellular microelectrode recording of postsynaptic potentials, we found DEX-induced inhibition of spontaneous and evoked neurotransmitter release as well as desynchronization of evoked exocytotic events in the mouse diaphragm neuromuscular junction. These effects were suppressed by SKF-86,466, a selective α2-AR antagonist. An activator of GIRK channels ML297 had the same effects on neurotransmitter release as DEX. By contrast, inhibition of GIRK channels with tertiapin-Q prevented the action of DEX on evoked neurotransmitter release, but not on spontaneous exocytosis. The synaptic vesicle exocytosis is strongly dependent on Ca2+ influx through voltage-gated Ca2+ channels (VGCCs), which can be negatively regulated via α2-AR - GIRK channel axis. Indeed, inhibition of P/Q-, L-, N- or R-type VGCCs prevented the inhibitory action of DEX on evoked neurotransmitter release; antagonists of P/Q- and N-type channels also suppressed the DEX-mediated desynchronization of evoked exocytotic events. Furthermore, inhibition of P/Q-, L- or N-type VGCCs precluded the frequency decrease of spontaneous exocytosis upon DEX application. Thus, α2-ARs acting via GIRK channels and VGCCs (mainly, P/Q- and N-types) exert inhibitory effect on the neuromuscular communication by attenuating and desynchronizing evoked exocytosis. In addition, α2-ARs can suppress spontaneous exocytosis through GIRK channel-independent, but VGCC-dependent pathway.

Sympathetic Innervation and Endogenous Catecholamines in Neuromuscular Preparations of Muscles with Different Functional Profiles.

Influence of the sympathetic nervous system on the work of skeletal muscles contractile apparatus is now beyond doubt. However, until recently there was no evidence that the endings of sympathetic nerves can be located in close proximity to the neuromuscular synapses, and there is also no reliable data on how much endogenous adrenaline and noradrenaline can be contained near the synaptic contact in skeletal muscles. In this research, using fluorescent analysis, immunohistochemical and enzyme immunoassays the isolated neuromuscular preparations of three skeletal muscles of different functional profiles and containing different types of muscle fibers were examined. Close contact between the sympathetic and motor cholinergic nerve endings and the presence of tyrosine hydroxylase in this area were demonstrated. Concentrations of endogenous adrenaline and noradrenaline in the solution perfusing the neuromuscular preparation were determined under different modes of its functioning. The effects of α and ß adrenoreceptor blockers on the processes of acetylcholine quantal secretion from the motor nerve endings were compared. The data obtained provide evidence for the presence of endogenous catecholamines in the neuromuscular junction region and their role in modulation of the synaptic function.

Catecholamine-dependent hyperpolarization of the junctional membrane via ß2- adrenoreceptor/Gi-protein/α2-Na-K-ATPase pathway.

We investigated the effects of catecholamines, adrenaline and noradrenaline, as well as ß-adrenoceptor (AR) modulators on a resting membrane potential at the junctional and extrajunctional regions of mouse fast-twitch Levator auris longus muscle. The aim of the study was to find which AR subtypes, signaling molecules and Na,K-ATPase isoforms are involved in the hyperpolarizing action of catecholamines and whether this action could be accompanied by changes in the pump abundance on the sarcolemma. Adrenaline, noradrenaline and specific ß2-AR agonist induced hyperpolarization of both junctional and extrajunctional membrane, but the underlying mechanisms were different. In the junctional membrane the hyperpolarization depended on α2 isoform of the Na,K-ATPase and Gi-protein, whereas in the extrajunctional regions the hyperpolarization mainly relied on α1 isoform of Na,K-ATPase and adenylyl cyclase activities. In both junctional and extrajunctional regions, AR activation caused an increase in Na,K-ATPase abundance in the plasmalemma in a protein kinase A-dependent manner. Thus, the compartment-specific mechanisms are responsible for catecholamine-mediated hyperpolarization in the skeletal muscle.

Presynaptic Acetylcholine Receptors Modulate the Time Course of Action Potential-Evoked Acetylcholine Quanta Secretion at Neuromuscular Junctions.

For effective transmission of excitation in neuromuscular junctions, the postsynaptic response amplitude must exceed a critical level of depolarization to trigger action potential spreading along the muscle-fiber membrane. At the presynaptic level, the end-plate potential amplitude depends not only on the acetylcholine quanta number released from the nerve terminals in response to the nerve impulse but also on a degree of synchronicity of quanta releases. The time course of stimulus-phasic synchronous quanta secretion is modulated by many extra- and intracellular factors. One of the pathways to regulate the neurosecretion kinetics of acetylcholine quanta is an activation of presynaptic autoreceptors. This review discusses the contribution of acetylcholine presynaptic receptors to the control of the kinetics of evoked acetylcholine release from nerve terminals at the neuromuscular junctions. The timing characteristics of neurotransmitter release is nowadays considered an essential factor determining the plasticity and efficacy of synaptic transmission.

Adrenergic receptors control frequency-dependent switching of the exocytosis mode between "full-collapse" and "kiss-and-run" in murine motor nerve terminal.

AIMS: Neurotransmitter release from the synaptic vesicles can occur through two modes of exocytosis: "full-collapse" or "kiss-and-run". Here we investigated how increasing the nerve activity and pharmacological stimulation of adrenoceptors can influence the mode of exocytosis in the motor nerve terminal. METHODS: Recording of endplate potentials with intracellular microelectrodes was used to estimate acetylcholine release. A fluorescent dye FM1-43 and its quenching with sulforhodamine 101 were utilized to visualize synaptic vesicle recycling. KEY FINDINGS: An increase in the frequency of stimulation led to a decrease in the rate of FM1-43 unloading despite the higher number of quanta released. High frequency activity promoted neurotransmitter release via the kiss-and-run mechanism. This was confirmed by experiments utilizing (I) FM1-43 dye quencher, that is able to pass into the synaptic vesicle via fusion pore, and (II) loading of FM1-43 by compensatory endocytosis. Noradrenaline and specific α2-adrenoreceptors agonist, dexmedetomidine, controlled the mode of synaptic vesicle recycling at high frequency activity. Their applications favored neurotransmitter release via full-collapse exocytosis rather than the kiss-and-run pathway. SIGNIFICANCE: At the diaphragm neuromuscular junctions, neuronal commands are translated into contractions necessary for respiration. During stress, an increase in discharge rate of the phrenic nerve shifts the exocytosis from the full-collapse to the kiss-and-run mode. The stress-related molecule, noradrenaline, restricts neurotransmitter release in response to a high frequency activity, and prevents the shift in the mode of exocytosis through α2-adrenoceptor activation. This may be a component of the mechanism that limits overstimulation of the respiratory system during stress.

Enhancement of the Temporal Resolution of Fluorescent Signals Acquired by the Confocal Microscope.

Here, we describe a method of acquisition of fast fluorescent signals with the help of the laser scanning confocal microscope (LSCM). Our method permits an increase in the temporal resolution of acquired signals. The method is based on LSCM recordings of fast fluorescent signals with the shortest achievable time sweep, which are performed with the help of a proprietary algorithm. A series of recordings is made in multiple steps; at each step, the fluorescent signal is incremented by a time interval smaller than the time sweep of the frame of LSCM. The size of the increment determines the achievable time resolution. The convolution of the recorded images results in a signal with the temporal resolution determined by the chosen time increment. This method was applied to register the change in fluorescence (calcium transient) of calcium dye preloaded into peripheral nerve endings by electrical stimulation of the motor nerve. Calculated parameters of the calcium transient were identical to the parameters obtained earlier with the help of a high-speed camera and photodiode. We conclude that the method described here can be applied for the registration of fast fluorescent signals by LSCM with a high spatial and temporal resolution.

Diverse Effects of Noradrenaline and Adrenaline on the Quantal Secretion of Acetylcholine at the Mouse Neuromuscular Junction.

Despite the long history of investigations of adrenergic compounds and their biological effects, specific mechanisms of their action in distinct compartments of the motor unit remain obscure. Recent results have suggested that not only skeletal muscles but also the neuromuscular junctions represent important targets for the action of catecholamines. In this paper, we describe the effects of adrenaline and noradrenaline on the frequency of miniature endplate potentials, the quantal content of the evoked endplate potentials and the kinetics of acetylcholine quantal release in the motor nerve endings of the mouse diaphragm. Noradrenaline and adrenaline decreased the frequency of the spontaneous release of acetylcholine quanta. The effect of noradrenaline was prevented by the ß adrenoreceptor blocker propranolol, whereas the action of adrenaline was abolished by the α adrenoreceptor antagonist phentolamine. Noradrenaline did not alter the quantal content of endplate potentials, while adrenaline suppressed the evoked release of acetylcholine. Blocking the α adrenoreceptors prevented the decrease in quantal secretion caused by adrenaline. Quantal release became more asynchronous under noradrenaline, as evidenced by a greater dispersion of real synaptic delays; in contrast, adrenaline synchronized the release process. Our data suggest an involvement of α and ß adrenoreceptors in the diverse modulation of the frequency of miniature endplate potentials, the quantal content of the evoked endplate potentials and the kinetics of acetylcholine quantal secretion in the mouse neuromuscular junction. Moreover, the adrenoblockers affected both the evoked and spontaneous quantal release of acetylcholine, suggesting the presence of endogenous catecholamines in the vicinity of cholinergic synapses.

Reorganization of Septins Modulates Synaptic Transmission at Neuromuscular Junctions.

Septins (Sept) are highly conserved Guanosine-5'-triphosphate (GTP)-binding cytoskeletal proteins involved in neuronal signaling in the central nervous system but their involvement in signal transmission in peripheral synapses remains unclear. Sept5 and Sept9 proteins were detected in mouse peripheral neuromuscular junctions by immunofluorescence with a greater degree of co-localization with presynaptic than postsynaptic membranes. Preincubation of neuromuscular junction preparations with the inhibitor of Sept dynamics, forchlorfenuron (FCF), decreased co-localization of Sept with presynaptic membranes. FCF introduced ex vivo or in vivo had no effect on the amplitude of the spontaneous endplate currents (EPCs), indicating the absence of postsynaptic effects of FCF. However, FCF decreased acetylcholine (ACh) quantal release in response to nerve stimulation, reduced the amplitude of evoked quantal currents and decreased the number of quanta with long synaptic delays, demonstrating the presynaptic action of FCF. Nevertheless, FCF had no effect on the amplitude of calcium transient in nerve terminals, as detected by calcium-sensitive dye, and slightly decreased the ratio of the second response amplitude to the first one in paired-pulse experiments. These results suggest that FCF-induced decrease in ACh quantal secretion is not due to a decrease in Ca2+ influx but is likely related to the impairment of later stages occurring after Ca2+ entry, such as trafficking, docking or membrane fusion of synaptic vesicles. Therefore, Sept9 and Sept5 are abundantly expressed in presynaptic membranes, and disruption of Sept dynamics suppresses the evoked synchronous and delayed asynchronous quantal release of ACh, strongly suggesting an important role of Sept in the regulation of neurotransmission in peripheral synapses.

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.

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.

Kinetics of acetylcholine quanta release at the neuromuscular junction during high-frequency nerve stimulation.

The effects of high-frequency nerve stimulation (10-100 Hz) on the kinetics of evoked acetylcholine quanta secretion from frog motor nerve endings were studied. The amplitude and temporal parameters of uni- and multiquantal endplate currents were analysed to estimate the possible changes in the degree of synchrony of quantal release. The frog neuromuscular synapse is unusually long and we have placed special emphasis on evaluating the velocity of propagation of excitation along the nonmyelinated nerve ending as this might influence the synchrony of release from the whole terminal and hence affect the time course of postsynaptic currents. The data show that high-frequency firing leads to the desynchronization of acetylcholine release from motor nerve endings governed by at least two independent factors, namely a reduction of nerve pulse propagation velocity in the nonmyelinated parts of the axon and a change of secretion kinetics at single active zones. A computer reconstruction of the multiquantal synaptic response was performed to estimate any contribution of each of the above factors to the total rate of release and amplitude and time characteristics of the endplate currents. The results indicate that modification of the kinetics of neurotransmitter quanta release during high-frequency firing should be taken into account when mechanisms underlying the plasticity of chemical synapses are under investigation.

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.