The role of calcium in modulation of the kinetics of synchronous and asynchronous quantal release at the neuromuscular junction

To elucidate the mechanisms of calcium regulation of the kinetics of the evoked neurotransmitter quantal release, we have investigated the temporal parameters of acetylcholine secretion in the mouse neuro-muscular junction at varying extracellular calcium concentration, in the presence of calcium channel blockers or intracellular calcium buffers. Acetylcholine secretion was induced by the motor nerve stimulation at a low frequency, which did not produce facilitation of the neurotransmitter release. The analysis of histograms of synaptic delays of uniquantal endplate currents recorded during 50 ms after the presynaptic action potential revealed three components of the secretion process: early and late periods of synchronous release and a delayed asynchronous release. At reduced extracellular calcium level, the relative number of quanta released during the asynchronous phase of secretion increased, while the rate of quantal release during the early synchronous period decreased. The findings support the hypothesis of participation of low- and high-affinity calcium sensors with different calcium binding kinetics in regulation of, respectively, synchronous and asynchronous release of neurotransmitter quanta.     

Presynaptic receptors regulating the time course of neurotransmitter release from vertebrate nerve endings

A number of different types of presynaptic receptors was revealed in central and peripheral chemical synapses activated both by main mediator and co-mediators released simultaneously. Physiological significance and mechanisms of functioning of these receptors are not clear yet. They are assumed to provide negative or positive feedback decreasing or increasing the number of neurotransmitter quanta released in response to nerve impulse and thus regulating synaptic transmission. At the same time, there is one more way of secretion process modulation associated with the changes of timing of transmitter release. This mechanism was shown to contribute to the efficiency of synaptic transmission. The role of presynaptic receptors in regulation of the kinetics of quanta release is one of the interesting questions of modern neurophysiology. This paper overviews the results obtained by the authors that demonstrate the contribution of presynaptic receptors of different types into the regulation of temporal parameters of quantal secretion at the vertebrates neuromuscular junction. It was shown that activation of the cholinergic nicotinic receptors leads to a decrease of the amplitude of postsynaptic response not only due to reduction of the quantity of released quanta but also due to increased the level of asynchronous release. On the contrary, the facilitating effect of catecholamines on the neuromuscular synapse is the result of activation of presynaptic β₁-adrenoreceptors which leads to greater synchronization of release process and, consequently, to the increase of the amplitude of the postsynaptic response. Presynaptic purine receptors, involved in the modulation the intensity of secretion, are also capable of alteration of the time course of secretion. Activation of ryanodine receptors results in the increase of the number of quanta released with prolonged latencies leading to appearance of the phase of delayed asynchronous neurotransmitter release.     

The Role of Endogenous Calcitonin Gene-Related Peptide in the Neurotransmitter Quantal Size Increase in Mouse Neuromuscular Junctions

Changes in parameters of spontaneous acetylcholine (ACh) quantal secretion caused by prolonged high-frequency burst activity of neuromuscular junctions and possible involvement of endogenous calcitonin gene-related peptide (CGRP) and its receptors in these changes were studied. With this purpose, miniature endplate potentials (MEPPs) were recorded using standard microelectrode technique in isolated neuromuscular preparations of m. EDL–n. peroneus after a prolonged high-frequency nerve stimulation (30 Hz for 2 min). An increase in the MEPP amplitudes and time course was observed in the postactivation period that reached maximum 20–30 min after nerve stimulation and progressively faded in the following 30 min of recording. Inhibition of vesicular ACh transporter with vesamicol (1 μM) fully prevented this “wave” of the MEPP enhancement. This indicates the presynaptic origin of the MEPP amplitude increase, possibly mediated via intensification of synaptic vesicle loading with ACh and subsequent increase of the quantal size. Competitive antagonist of the CGRP receptor, truncated peptide isoform CGRP_8–37 (1 μM), had no effect on spontaneous secretion parameters by itself but was able to prevent the appearance of enhanced MEPPs in the postactivation period. This suggests the involvement of endogenous CGRP and its receptors in the observed MEPP enhancement after an intensive nerve stimulation. Ryanodine in high concentration (1 μM) that blocks ryanodine receptors and stored calcium release did not influence spontaneous ACh secretion but prevented the increase of the MEPP parameters in the postactivation period. Altogether, the data indicate that an intensive nerve stimulation, which activates neuromuscular junctions and muscle contractions, leads to a release of endogenous CGRP into synaptic cleft and this release strongly depends on the efflux of stored calcium. The released endogenous CGRP is able to exert an acute presynaptic effect on nerve terminals, which involves its specific receptor action and intracellular cascades leading to intensification of ACh loading into synaptic vesicles and an increase in the ACh quantal size.     

Changes in the kinetics of quanta secretion-effective mechanism of synaptic transmission modulation

It is widely accepted that the leading presynaptic mechanisms underlying the synaptic plasticity involve changes of the number of neurotransmitter quanta released by one nerve pulse (the quantal content of postsynaptic response) and of the size of a single quantum. In addition, the existence of one more effective though previously ignored mechanism of modulation of synaptic plasticity was suggested related to the change in the time course (kinetics) of secretion of single neurotransmitter quanta forming the multiquantal response. This article reviews current data (including the authors' own results) on the kinetics of evoked neurotransmitter quanta secretion from motor nerve endings in peripheral synapses, mechanisms of their modulation and methods of quantitative analysis.     

In vivo regulation of acetylcholinesterase insertion at the neuromuscular junction

The efficiency of synaptic transmission between nerve and muscle depends on the number and density of acetylcholinesterase molecules (AChE) at the neuromuscular junction. However, little is known about the way this density is maintained and regulated in vivo. By using time lapse and quantitative fluorescence imaging assays in living mice, we demonstrated that insertion of new AChEs occurs within hours of saturating pre-existing AChEs with fasciculin2, a snake toxin that selectively labels AChE. In the absence of muscle postsynaptic activity or evoked nerve presynaptic neurotransmitter release, AChE insertion was decreased significantly, whereas direct stimulation of the muscle completely restored AChE insertion to control levels. This activity-dependent AChE insertion is mediated by intracellular calcium. In muscle stimulated in the presence of a Ca2+ channel blocker or calcium-permeable Ca2+ chelator, AChE insertion into synapses was significantly decreased, whereas ryanodine or ionophore A12387 treatment of blocked and unstimulated synapses significantly increased AChE insertion. These results demonstrated that synaptic activity is critical for AChE insertion and indicated that a rise in intracellular calcium either through voltage-gated calcium channels or from intracellular stores is critical for proper AChE insertion into the adult synapse.     

Facilitation and Depression of Neuromuscular Transmission under Conditions of Blockade of Ryanodine Receptors

In our experiments on mice, end-plate currents (EPC) evoked by stimulation of the phrenic nerve were intracellularly recorded in neuromuscular synaptic junctions of the phrenic muscle. We studied the effects of a specific blocker of ryanodine receptors, ryanodine (10 to 20 M), on the amplitude and time parameters of EPC under conditions of tetanic facilitation and depression of synaptic transmission at frequencies of stimulation of 4 to 200 sec^-1. Ryanodine inhibited facilitation at stimulation frequencies of 7 to 70 sec^-1 (with maximum effect at 20 sec^-1) and accelerated depression. In the presence of ryanodine, an initial rundown of the EPC amplitude in the course of depression of transmission increased at high frequencies of stimulation (50 to 100 sec^-1), whereas the EPC amplitude at the plateau level decreased already at low frequencies (4 to 7 sec^-1). We concluded that the changes in facilitation and depression resulted from blocking of the presynaptic ryanodine receptors by ryanodine. It seems probable that calcium release from the calcium stores in murine motor terminals is a factor involved in the control of processes of transmitter secretion during short-term rhythmic activation of the junction.     

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).     

Quantum Characteristics of Neurotransmitter Release in GABA-ergic Synapses of Cultured Neurons of the Rat Spinal Cord

Using the whole-cell patch-clamp technique and stimulation of a single presynaptic terminal, we studied peculiarities of GABA release in inhibitory synapses of cultured neurons of the rat spinal cord. Analyzing the amplitude distributions of evoked inhibitory postsynaptic currents, we estimated the main quantum parameters of transmitter release. It was demonstrated that the minimum transmitter release in GABA-ergic synapses of spinal neurons cultured 9 to 11 days is multiquantum (packets containing at least 2 or 3 quanta). The distribution of the number of released quanta sufficiently agreed with that theoretically calculated according to the Poisson law. It is hypothesized that the minimum simultaneous two (three-)-quantum release of GABA in synapses of spinal neurons can be related to synchronous involvement of two closely adjacent excited terminals, each of which possesses one active zone, or of one terminal with two active zones.     

Presynaptic kainate and NMDA receptors are implicated in the modulation of GABA release from cortical and hippocampal nerve terminals

One of the pathways implicated in a fine-tuning control of synaptic transmission is activation of the receptors located at the presynaptic terminal. Here we investigated the intracellular events in rat brain cortical and hippocampal nerve terminals occurring under the activation of presynaptic glutamate receptors by exogenous glutamate and specific agonists of ionotropic receptors, NMDA and kainate. Involvement of synaptic vesicles in exocytotic process was assessed using [³H]GABA and pH-sensitive fluorescent dye acridine orange (AO). Glutamate as well as NMDA and kainate were revealed to induce [³H]GABA release that was not blocked by NO-711, a selective blocker of GABA transporters. AO-loaded nerve terminals responded to glutamate application by the development of a two-phase process. The first phase, a fluorescence transient completed in ∼1 min, was similar to the response to high K⁺. It was highly sensitive to extracellular Ca²⁺ and was decreased in the presence of the NMDA receptor antagonist, MK-801. The second phase, a long-lasting process, was absolutely dependent on extracellular Na⁺ and attenuated in the presence of CNQX, the kainate receptor antagonist. NMDA as well as kainate per se caused a rapid and abrupt neurosecretory process confirming that both glutamate receptors, NMDA and kainate, are involved in the control of neurotransmitter release. It could be suggested that at least two types ionotropic receptor are attributed to glutamate-induced two-phase process, which appears to reflect a rapid synchronous and a more prolonged asynchronous vesicle fusion.     

Functional coupling of Ca(2+) channels to ryanodine receptors at presynaptic terminals. Amplification of exocytosis and plasticity

Ca(2+)-induced Ca(2+) release (CICR) enhances a variety of cellular Ca(2+) signaling and functions. How CICR affects impulse-evoked transmitter release is unknown. At frog motor nerve terminals, repetitive Ca(2+) entries slowly prime and subsequently activate the mechanism of CICR via ryanodine receptors and asynchronous exocytosis of transmitters. Further Ca(2+) entry inactivates the CICR mechanism and the absence of Ca(2+) entry for >1 min results in its slow depriming. We now report here that the activation of this unique CICR markedly enhances impulse-evoked exocytosis of transmitter. The conditioning nerve stimulation (10-20 Hz, 2-10 min) that primes the CICR mechanism produced the marked enhancement of the amplitude and quantal content of end-plate potentials (EPPs) that decayed double exponentially with time constants of 1.85 and 10 min. The enhancement was blocked by inhibitors of ryanodine receptors and was accompanied by a slight prolongation of the peak times of EPP and the end-plate currents estimated from deconvolution of EPP. The conditioning nerve stimulation also enhanced single impulse- and tetanus-induced rises in intracellular Ca(2+) in the terminals with little change in time course. There was no change in the rate of growth of the amplitudes of EPPs in a short train after the conditioning stimulation. On the other hand, the augmentation and potentiation of EPP were enhanced, and then decreased in parallel with changes in intraterminal Ca(2+) during repetition of tetani. The results suggest that ryanodine receptors exist close to voltage-gated Ca(2+) channels in the presynaptic terminals and amplify the impulse-evoked exocytosis and its plasticity via CICR after Ca(2+)-dependent priming.     

Rhythmic Activity of Murine Neuromuscular Junctions upon Activation of Release of Stored Calcium in the Presence of a Calcium Buffer

Using a two-electrode voltage-clamp technique, we recorded end-plate currents (EPCs) in neuromuscular synaptic junctions of the murine diaphragm upon rhythmic stimulation of the n. phrenicus with frequencies of 7, 20, 50, 70, and 100 sec⁻¹. Parameters of EPC series were analyzed against the background of the action of a mobilizer of intracellular calcium, ryanodine (0.5 μM), after the loading of terminals by 1.2 mM BAPTA (calcium buffer with rapid dynamics of binding of calcium), and upon the action of ryanodine in the presence of BAPTA. Under the action of ryanodine, the amplitude and quantum content of EPC within the plateau phase increased by 100 to 150% (P < 0.05). Loading with BAPTA evoked sharp decreases in the quantum content of unitary EPCs, the intensity of the initial facilitation, and the level of the EPC plateau in series within the entire range of stimulation frequencies used. Against the background of the action of BAPTA, the facilitatory effect of ryanodine increased; inhibitory effects of BAPTA with respect to the amplitude of unitary EPC and the level of the initial facilitation were completely compensated, whereas the level of EPC at the plateau stage increased to levels exceeding the control values by 50 to 70%. The ability of ryanodine to facilitate the transmitter (acetylcholine) release, which was enhanced in the presence of BAPTA, was completely neutralized by a blocker of L-type calcium channels, verapamil (5 μM). In the absence of BAPTA, verapamil did not influence the effects of ryanodine. We hypothesize that in the presence of BAPTA calcium channels of L type whose activity is resistive to the buffer action of BAPTA are disinhibited. The calcium current through L-type channels, perhaps, is capable of stimulating calcium release from the stores of nerve terminals and, as a consequence, of intensifying the facilitatory effect of ryanodine on the release of acetylcholine. After verapamil-induced blockade of this current, BAPTA demonstrates the ability to prevent the facilitatory effect of ryanodine on the transmitter release. Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 330–338, July–August, 2005.     

Presynaptic calcium signals during neurotransmitter release: detection with fluorescent indicators and other calcium chelators.

Synthetic calcium buffers, including fluorescent calcium indicators, were microinjected into squid 'giant' presynaptic nerve terminals to investigate the calcium signal that triggers neurotransmitter secretion. Digital imaging methods, applied in conjunction with the fluorescent calcium indicator dye fura-2, reveal that transient rises in presynaptic calcium concentration are associated with action potentials. Transmitter release terminates within 1-2 ms after a train of action potentials, even though presynaptic calcium concentration remains at micromolar levels for many seconds longer. Microinjection of the calcium buffer, EGTA, into the presynaptic terminal has no effect on transmitter release evoked by single presynaptic action potentials. EGTA injection does, however, block the change in calcium concentration measured by fura-2. Therefore, the calcium signal measured by fura-2 is not responsible for triggering release. These results suggest that the rise in presynaptic calcium concentration that triggers release must be highly localized to escape detection with fura-2 imaging. Unlike EGTA, microinjection of BAPTA--a calcium buffer with an equilibrium affinity for calcium similar to that of EGTA--produces a potent, dose-dependent, and reversible block of action-potential evoked transmitter release. The superior ability of BAPTA to block transmitter release apparently is due to the more rapid calcium-binding kinetics of BAPTA compared to EGTA. Because EGTA should bind calcium within a few tens of microseconds under the conditions of our experiments, the inability of EGTA to block release indicates that transmitter release is triggered within a few tens of microseconds after the entry of calcium into the presynaptic terminal.(ABSTRACT TRUNCATED AT 250 WORDS)     

The actin cytoskeleton and neurotransmitter release: an overview

Here we review evidence that actin and its binding partners are involved in the release of neurotransmitters at synapses. The spatial and temporal characteristics of neurotransmitter release are determined by the distribution of synaptic vesicles at the active zones, presynaptic sites of secretion. Synaptic vesicles accumulate near active zones in a readily releasable pool that is docked at the plasma membrane and ready to fuse in response to calcium entry and a secondary, reserve pool that is in the interior of the presynaptic terminal. A network of actin filaments associated with synaptic vesicles might play an important role in maintaining synaptic vesicles within the reserve pool. Actin and myosin also have been implicated in the translocation of vesicles from the reserve pool to the presynaptic plasma membrane. Refilling of the readily releasable vesicle pool during intense stimulation of neurotransmitter release also implicates synapsins as reversible links between synaptic vesicles and actin filaments. The diversity of actin binding partners in nerve terminals suggests that actin might have presynaptic functions beyond synaptic vesicle tethering or movement. Because most of these actin-binding proteins are regulated by calcium, actin might be a pivotal participant in calcium signaling inside presynaptic nerve terminals. However, there is no evidence that actin participates in fusion of synaptic vesicles.     

Calcium signaling mechanisms in the gastric parietal cell.

Gastric hydrochloric acid (HCl) secretion is stimulated in vivo by histamine, acetylcholine, and gastrin. In vitro studies have shown that histamine acts mainly via a cAMP-dependent pathway, and acetylcholine acts via a calcium-dependent pathway. Histamine also elevates intracellular calcium ([Ca2+]i) in parietal cells. Both gastrin and acetylcholine release histamine from histamine-containing cells. In humans, rats, and rabbits, there is considerable controversy as to whether or not gastrin receptors are also present on the parietal cell. We utilized digitized video image analysis techniques in this study to demonstrate gastrin-induced changes in intracellular calcium in single parietal cells from rabbit in primary culture. Gastrin also stimulated a small increase in [14C]-aminopyrine (AP) accumulation, an index of acid secretory responsiveness in cultured parietal cells. In contrast to histamine and the cholinergic agonist, carbachol, stimulation of parietal cells with gastrin led to rapid loss of the calcium signaling response, an event that is presumed to be closely related to gastrin receptor activation. Moreover, different calcium signaling patterns were observed for histamine, carbachol, and gastrin, Previous observations coupled with present studies using manganese, caffeine, and ryanodine suggest that agonist-stimulated increases in calcium influx into parietal cells do not occur via voltage-sensitive calcium channels or nonspecific divalent cation channels. It also appears to be unlikely that release of intracellular calcium is mediated by a muscle or neuronal-type ryanodine receptor. We hypothesize that calcium influx may be mediated by either a calcium exchange mechanism or by an unidentified calcium channel subtype that possesses different molecular characteristics as compared to muscle, nerve, and certain secretory cell types such as, for example, the adrenal chromaffin cell. Release of intracellular calcium may be mediated via both InsP3-sensitive and -insensitive mechanisms. The InsP3-insensitive calcium pools, if present, do not appear, however, to possess ryanodine receptors capable of modulating calcium efflux from these storage sites.     

Multitude of ion channels in the regulation of transmitter release

The presynaptic nerve terminal is of key importance in communication in the nervous system. Its primary role is to release transmitter quanta on the arrival of an appropriate stimulus. The structural basis of these transmitter quanta are the synaptic vesicles that fuse with the surface membrane of the nerve terminal, to release their content of neurotransmitter molecules and other vesicular components. We subdivide the control of quantal release into two major classes: the processes that take place before the fusion of the synaptic vesicle with the surface membrane (the pre-fusion control) and the processes that occur after the fusion of the vesicle (the post-fusion control). The pre-fusion control is the main determinant of transmitter release. It is achieved by a wide variety of cellular components, among them the ion channels. There are reports of several hundred different ion channel molecules at the surface membrane of the nerve terminal, that for convenience can be grouped into eight major categories. They are the voltage-dependent calcium channels, the potassium channels, the calcium-gated potassium channels, the sodium channels, the chloride channels, the non-selective channels, the ligand gated channels and the stretch-activated channels. There are several categories of intracellular channels in the mitochondria, endoplasmic reticulum and the synaptic vesicles. We speculate that the vesicle channels may be of an importance in the post-fusion control of transmitter release.     

Autonomous function of synaptotagmin 1 in triggering synchronous release independent of asynchronous release

Ca(2+) triggers neurotransmitter release in at least two principal modes, synchronous and asynchronous release. Synaptotagmin 1 functions as a Ca(2+) sensor for synchronous release, but its role in asynchronous release remains unclear. We now show that in cultured cortical neurons stimulated at low frequency (or Hz), deletion of synaptotagmin 1 also alters only synchronous, not asynchronous, release during the stimulus train, but dramatically enhances "delayed asynchronous release" following the stimulus train. Thus synaptotagmin 1 functions as an autonomous Ca(2+) sensor independent of asynchronous release during isolated action potentials and action potential trains, but restricts asynchronous release induced by residual Ca(2+) after action potential trains. We propose that synaptotagmin 1 occupies release "slots" at the active zone, possibly in a Ca(2+)-independent complex with SNARE proteins that are freed when action potential-induced Ca(2+) influx activates synaptotagmin 1.     

The time course of the evoked secretion of the mediator quanta in various regions of the frog motor nerve ending

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.     

A buffering model for calcium-dependent neurotransmitter release

A simple model is proposed, whereby a single buffering system for intracellular calcium accounts for the steep external Ca dependence of neurotransmitter release during depolarization of the presynaptic nerve terminal. Ca entry and buffering in the nerve terminal are assumed to be saturable; release is assumed to be proportional to intracellular Ca. The novel feature of this model is that it explains the apparent cooperative relationship between transmitter release and extracellular calcium, without invoking cooperative Ca binding.     

The effect of calcium ions on the secretion of quanta evoked by an impulse at nerve terminal release sites

A study has been made of the effects of calcium ions on the number of quanta secreted from all the release sites at an amphibian motor nerve terminal recorded with an intracellular microelectrode (m) compared with the number secreted simultaneously from a small number of release sites recorded with an extracellular microelectrode (me). If the endplate potential was made subthreshold by lowering the external calcium concentration ([Ca]o less than or equal to 0.4 mM), it was possible to find small groups of release sites for which me was comparable to m, indicating considerable nonuniformity in the probability of release of a quantum at different groups of release sites (Pe) in a given [Ca]o. Increasing [Ca]o in the range from 0.25 to 0.4 mM increased the probability of release of a quantum at groups of release sites (Pe), independent of the initial value of Pe, and the dependence of Pe on [Ca]o followed a fourth power relationship. A conditioning impulse enhanced the probability of release of a quantum by a subsequent test impulse at release sites, if Pe was less than 1.0 during the conditioning impulse. It is shown that the present observations regarding the dependence of Pe on [Ca]o and on conditioning impulses can be quantitatively predicted from previous observations regarding the dependence of the binomial parameters m, p, and n on [Ca]o and on conditioning impulses determined with intracellular electrodes, if the probability of secretion of a quantum at a release site (Pj) is different for different release sites and Pj is distributed as a beta random variable.     

Caffeine-induced modulation of network oscillation in a molluscan olfactory center

Calcium release from intracellular stores has various actions in neurons, but its effects on network oscillation have not been well understood. The olfactory center (procerebrum, PC) of the terrestrial slug Limax valentianus shows a regular oscillation in the local field potential (LFP). Here we report that caffeine, which is an agonist for ryanodine receptors and triggers calcium release from intra-cellular stores, has strong modulatory effects on the PC. In isolated PC neurons, caffeine enhanced the cytoplasmic calcium concentration, and this was blocked by ryanodine. Caffeine elevated the frequency and amplitude of the LFP oscillation, which was also blocked by ryanodine. The time lag between the frequency and amplitude effects suggests distinct mechanisms for the modulation of these two parameters. These results suggest that calcium release from intracellular stores through ryanodine receptors activates network activity in the PC.