The unitary evoked potential at the frog nerve-muscle junction results from synchronous gating of fusion pores at docked vesicles

Exocytosis of a single vesicle has been proposed as the mechanism which determines quantal size by releasing a prepackaged and standard amount of acetylcholine. As first described by del Castillo and Katz (1954) the endplate potential is composed of 100 unitary events and the small variance suggests a binomial release from 100 "discrete patches of membrane". However, exocytosis of 100 vesicles selected randomly from 5000 docked vesicles would yield a variance that is 7 times greater than observed values. We propose that the presynaptic ridge with its compliment of docked vesicles functions as the "discrete patch of membrane" such that arrays of calcium activated fusion pores meter transmitter to form the unit of release. A model based on the synchronous flicker of a large number of fusion pores produces the small variance of both miniature end plate potentials and unitary end plate potentials. Release from a single locus (fusion pore) would generate the sub-MEPP. This model permits vesicle trafficking and vesicular content depletion during tetanic stimulation and explains the frequency dependency of MEPP amplitudes and changes in sub-MEPP to bell-MEPP class ratios.     

The coupling between synaptic vesicles and Ca2+ channels determines fast neurotransmitter release

In order to release neurotransmitter synchronously in response to a presynaptic action potential, synaptic vesicles must be both release competent and located close to presynaptic Ca2+ channels. It has not been shown, however, which of the two is the more decisive factor. We tested this issue at the calyx of Held synapse by combining Ca2+ uncaging and electrophysiological measurements of postsynaptic responses. After depletion of the synaptic vesicles that are responsible for synchronous release during action potentials, uniform elevation of intracellular Ca2+ by Ca2+ uncaging could still elicit rapid release. The Ca2+ sensitivity of remaining vesicles was reduced no more than 2-fold, which is insufficient to explain the slow-down of the kinetics of release (10-fold) observed during a depolarizing pulse. We conclude that recruitment of synaptic vesicles to sites where Ca2+ channels cluster, rather than fusion competence, is a limiting step for rapid neurotransmitter release in response to presynaptic action potentials.     

The synaptic bouton acts like a slat shaker

The physiological quantal responses at the neuromuscular junction and the bouton-neuron show two classes based on amplitude such that the larger class is about 10 times that of the smaller class; and, the larger class is composed of the smaller class. The ratio of the two classes changes with synaptogenesis, degeneration, nerve stimulation, and is readily altered with various challenges (ionic, tonicity, pharmacological agents). Statistical analyses demonstrate that each bouton or release site at the neruomuscular junction (NMJ) secretes a standard amount of transmitter (one quantum) with each action potential. The amount of transmitter secreted (quantal size) is frequency dependent. The quantal-vesicular-exocytotic (QVE) hypothesis posits that the packet of secreted transmitter is released from one vesicle by exocytosis. The QVE hypothesis neither explains two quantal classes and subunits nor exocytosis of only one vesicle at each site. The latter observation requires a mechanism to select one vesicle from each array. Our porocytosis hypothesis states that the quantal packet is pulsed from an array of secretory pores. A salt shaker delivers a standard pinch of salt with each shake because salt flows through all openings in the cap. The variation in the pinch of salt or transmitter decreases with an increase in array size. The docked vesicles, paravesicular matrix, and porosomes (pores) of a release site form the secretory unit. In analogy with the sacromere as the functional unit of skeletal muscle, we term the array of docked vesicles and paravesicular grid along with the array of postsynaptic receptors a synaptomere. Pulsed secretion from an array explains the substructure of the postsynaptic response (quantum). The array guarantees a constant amount of secretion with each action potential and permits a given synapse to function in different responses because different frequencies would secrete signature amounts of transmitter. Our porocytosis hypothesis readily explains a change in quantal size during learning and memory with an increase in the number of elements (docked vesicles) composing the array.     

Ectopic release of synaptic vesicles

Exocytosis of synaptic vesicles is generally assumed to occur only at ultrastructurally defined presynaptic active zones. If release is restricted to these sites, receptors not located within the synaptic cleft must be activated by transmitter that diffuses out of the cleft or not be activated at all. Here we report that AMPA receptor-mediated quantal events resulting from climbing fiber release are observed in Bergmann glial cells in the cerebellar cortex. These quantal events are not coincident with quanta recorded in neighboring Purkinje cells which receive input from the same climbing fiber. As Bergmann glial membranes are excluded from the synaptic cleft, we propose that exocytosis can occur from climbing fiber release sites located directly across from Bergmann glial membranes. Such ectopic release may account for the majority of the Bergmann glial AMPA response evoked by climbing fiber stimulation.     

Neurotransmitter release at rapid synapses

The classical concept of the vesicular hypothesis for acetylcholine (ACh) release, one quantum resulting from exocytosis of one vesicle, is becoming more complicated than initially thought. 1) synaptic vesicles do contain ACh, but the cytoplasmic pool of ACh is the first to be used and renewed on stimulation. 2) The vesicles store not only ACh, but also ATP and Ca(2+) and they are critically involved in determining the local Ca(2+) microdomains which trigger and control release. 3) The number of exocytosis pits does increase in the membrane upon nerve stimulation, but in most cases exocytosis happens after the precise time of release, while it is a change affecting intramembrane particles which reflects more faithfully the release kinetics. 4) The SNARE proteins, which dock vesicles close to Ca(2+) channels, are essential for the excitation-release coupling, but quantal release persists when the SNAREs are inactivated or absent. 5) The quantum size is identical at the neuromuscular and nerve-electroplaque junctions, but the volume of a synaptic vesicle is eight times larger in electric organ; at this synapse there is enough ACh in a single vesicle to generate 15-25 large quanta, or 150-200 subquanta. These contradictions may be only apparent and can be resolved if one takes into account that an integral plasmalemmal protein can support the formation of ACh quanta. Such a protein has been isolated, characterised and called mediatophore. Mediatophore has been localised at the active zones of presynaptic nerve terminals. It is able to release ACh with the expected Ca(2+)-dependency and quantal character, as demonstrated using mediatophore-transfected cells and other reconstituted systems. Mediatophore is believed to work like a pore protein, the regulation of which is in turn likely to depend on the SNARE-vesicle docking apparatus.     

Brefeldin A increases the quantal size and alters the kinetics of catecholamine release from rat adrenal chromaffin cells.

The fungal metabolite, brefeldin A (BFA), is known to inhibit guanine nucleotide exchange on the ADP-ribosylating factors that are involved in vesicle membrane trafficking. Here, we investigated the action of BFA on Ca2+-regulated exocytosis in single rat adrenal chromaffin cells. Incubation of chromaffin cells with BFA (1 or 10 microM) for 2 h effectively disrupted the Golgi membranes but did not affect the pattern of catecholamine release triggered by high extracellular K+, which was monitored with carbon fiber amperometry along with cytosolic Ca2+ measurement. The BFA treatment, however, increased the mean quantal size of catecholamine-containing vesicles and the occurrence of amperometric events with a "foot" or "stand alone" signal (which reflects sluggish or incomplete dilation of the fusion pore). To examine whether BFA altered the Ca2+-dependence of exocytosis, we employed the whole-cell recording technique in conjunction with the capacitance measurement to measure exocytosis evoked from the entire cell during voltage-gated Ca2+ entry. Our results suggested that BFA treatment did not alter either the initial rate of capacitance increase or the total amount of capacitance increase. Therefore, in chromaffin cells, BFA treatment affects Ca2+-regulated exocytosis predominantly by increasing the quantal size and by slowing the fusion kinetics of some vesicles.     

The synaptic bouton acts like a salt shaker

The physiological quantal responses at the neuromuscular junction and the bouton-neuron show two classes based on amplitude such that the larger class is about 10 times that of the smaller class; and, the larger class is composed of the smaller class. The ratio of the two classes changes with synaptogenesis, degeneration, nerve stimulation, and is readily altered with various challenges (ionic, tonicity, pharmacological agents). Statistical analyses demonstrate that each bouton or release site at the neruomuscular junction (NMJ) secretes a standard amount of transmitter (one quantum) with each action potential. The amount of transmitter secreted (quantal size) is frequency dependent. The quantal-vesicular-exocytotic (QVE) hypothesis posits that the packet of secreted transmitter is released from one vesicle by exocytosis. The QVE hypothesis neither explains two quantal classes and subunits nor exocytosis of only one vesicle at each site. The latter observation requires a mechanism to select one vesicle from each array. Our porocytosis hypothesis states that the quantal packet is pulsed from an array of secretory pores. A salt shaker delivers a standard pinch of salt with each shake because salt flows through all openings in the cap. The variation in the pinch of salt or transmitter decreases with an increase in array size. The docked vesicles, paravesicular matrix, and porosomes (pores) of a release site form the secretory unit. In analogy with the sacromere as the functional unit of skeletal muscle, we term the array of docked vesicles and paravesicular grid along with the array of postsynaptic receptors a synaptomere. Pulsed secretion from an array explains the substructure of the postsynaptic response (quantum). The array guarantees a constant amount of secretion with each action potential and permits a given synapse to function in different responses because different frequencies would secrete signature amounts of transmitter. Our porocytosis hypothesis readily explains a change in quantal size during learning and memory with an increase in the number of elements (docked vesicles) composing the array.     

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

The Ca²⁺-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.     

The molecular mechanisms of quantum mediator secretion in the synapse

The modern condition of knowledge about the molecular mechanisms underlying the quantal transmitter release in the central and the peripheric synapses is analysed. The data about the synaptic vesicles types, their forming, transporting to the sites of release at the nerve endings, exo- and endocytosis processes are presented. Ultrastructural and molecular organization of active zone of nerve ending and transmitter release morphofunctional unit--secretosome, which includes synaptic vesicle, exocytosis protein complex and calcium channels, are described. The basic proteins involved in the exo- and endocytosis and their interactions during transmitter release are examined. The role of the intracellular buffer systems, calcium micro- and macrodomains in the quantal transmitter secretion are considered. The reasons of the active zones functional non-uniformity and plasticity and factors reduced transmitter release in the active zone to the single quantum are analysed.     

Probabilistic secretion of quanta: spontaneous release at active zones of varicosities, boutons, and endplates.

The amplitude-frequency histogram of spontaneous miniature endplate potentials follows a Gaussian distribution at mature endplates. This distribution gives the mean and variance of the quantum of transmitter. According to the vesicle hypothesis, this quantum is due to exocytosis of the contents of a single synaptic vesicle. Multimodal amplitude-frequency histograms are observed in varying degrees at developing endplates and at peripheral and central synapses, each of which has a specific active zone structure. These multimodal histograms may be due to the near synchronous exocytosis of more than one vesicle. In the present work, a theoretical treatment is given of the rise of intraterminal calcium after the stochastic opening of a calcium channel within a particular active zone geometry. The stochastic interaction of this calcium with the vesicle-associated proteins involved in exocytosis is then used to calculate the probability of quantal secretions from one or several vesicles at each active zone type. It is shown that this procedure can account for multiquantal spontaneous release that may occur at varicosities and boutons, compared with that at the active zones of motor nerve terminals.     

Independent Regulation of Basal Neurotransmitter Release Efficacy by Variable Ca2+ Influx and Bouton Size at Small Central Synapses

The efficacy of action potential evoked neurotransmitter release varies widely even among synapses supplied by the same axon, and the number of release-ready vesicles at each synapse is a major determinant of this heterogeneity. Here we identify a second, equally important, mechanism for release heterogeneity at small hippocampal synapses, the inter-synaptic variation of the exocytosis probability of release-ready vesicles. Using concurrent measurements of vesicular pool sizes, vesicular exocytosis rates, and presynaptic Ca²⁺ dynamics, in the same small hippocampal boutons, we show that the average fusion probability of release-ready vesicles varies among synapses supplied by the same axon with the size of the spike-evoked Ca²⁺ concentration transient. We further show that synapses with a high vesicular release probability exhibit a lower Ca²⁺ cooperativity, arguing that this is a direct consequence of increased Ca²⁺ influx at the active zone. We conclude that variability of neurotransmitter release under basal conditions at small central synapses is accounted for not only by the number of release-ready vesicles, but also by their fusion probabilities, which are set independently of bouton size by variable spike-evoked presynaptic Ca²⁺ influx.

Author Summary

Synaptic transmission underlies information transfer among neurons in the brain. The probability that a synapse will release neurotransmitter in response to an action potential varies widely, even among synapses supplied by the same axon. The molecular mechanisms underlying this heterogeneity remain poorly understood. At the level of single synapses, release efficacy is determined largely by two factors: (i) the number of neurotransmitter-containing vesicles ready to be released, and (ii) by the fusion probabilities of these vesicles. By using novel imaging techniques at individual hippocampal presynaptic boutons in culture, we distinguish two independent sources of variability of release probability in small central synapses. First, we find differences in the number of releasable vesicles, and second, we find differences in the exocytosis probability of individual vesicles. To our knowledge, this is the first direct experimental demonstration that the fusion probability of release-ready vesicles is variable among synapses supplied by a single axon, and contributes roughly as much to the overall variability in release probability as does the number of release-ready vesicles.     

A single vesicular glutamate transporter is sufficient to fill a synaptic vesicle

Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size.     

The probability of quantal secretion near a single calcium channel of an active zone

A Monte Carlo analysis has been made of calcium dynamics and quantal secretion at microdomains in which the calcium reaches very high concentrations over distances of <50 nm from a channel and for which calcium dynamics are dominated by diffusion. The kinetics of calcium ions in microdomains due to either the spontaneous or evoked opening of a calcium channel, both of which are stochastic events, are described in the presence of endogenous fixed and mobile buffers. Fluctuations in the number of calcium ions within 50 nm of a channel are considerable, with the standard deviation about half the mean. Within 10 nm of a channel these numbers of ions can give rise to calcium concentrations of the order of 100 microM. The temporal changes in free calcium and calcium bound to different affinity indicators in the volume of an entire varicosity or bouton following the opening of a single channel are also determined. A Monte Carlo analysis is also presented of how the dynamics of calcium ions at active zones, after the arrival of an action potential and the stochastic opening of a calcium channel, determine the probability of exocytosis from docked vesicles near the channel. The synaptic vesicles in active zones are found docked in a complex with their calcium-sensor associated proteins and a voltage-sensitive calcium channel, forming a secretory unit. The probability of quantal secretion from an isolated secretory unit has been determined for different distances of an open calcium channel from the calcium sensor within an individual unit: a threefold decrease in the probability of secretion of a quantum occurs with a doubling of the distance from 25 to 50 nm. The Monte Carlo analysis also shows that the probability of secretion of a quantum is most sensitive to the size of the single-channel current compared with its sensitivity to either the binding rates of the sites on the calcium-sensor protein or to the number of these sites that must bind a calcium ion to trigger exocytosis of a vesicle.     

Neurotransmitter release: fusion or 'kiss-and-run'?

The clear synaptic vesicles of neurons release their contents at the presynaptic membrane and are then quickly retrieved. However, it is unclear whether a complete cycle of exocytosis and endocytosis is always involved or whether neurotransmitter can be released by a transient interaction. Recent findings in chromaffin and mast cells suggest that exocytosis is preceded by the formation of a pore that has similar conductance properties to ion channels. The content of the secretory organelle partially escapes at this early step, but the pore can close before the vesicle fuses fully. This article looks at the evidence that quantal release of neurotransmitter from clear synaptic vesicles may occur by a similar 'kiss-and-run' mechanism.     

Mechanisms of synaptic vesicle recycling illuminated by fluorescent dyes

The recycling of synaptic vesicles in nerve terminals involves multiple steps, underlies all aspects of synaptic transmission, and is a key to understanding the basis of synaptic plasticity. The development of styryl dyes as fluorescent molecules that label recycling synaptic vesicles has revolutionized the way in which synaptic vesicle recycling can be investigated, by allowing an examination of processes in neurons that have long been inaccessible. In this review, we evaluate the major aspects of synaptic vesicle recycling that have been revealed and advanced by studies with styryl dyes, focussing upon synaptic vesicle fusion, retrieval, and trafficking. The greatest impact of styryl dyes has been to allow the routine visualization of endocytosis in central nerve terminals for the first time. This has revealed the kinetics of endocytosis, its underlying sequential steps, and its regulation by Ca2+. In studies of exocytosis, styryl dyes have helped distinguish between different modes of vesicle fusion, provided direct support for the quantal nature of exocytosis and endocytosis, and revealed how the probability of exocytosis varies enormously from one nerve terminal to another. Synaptic vesicle labelling with styryl dyes has helped our understanding of vesicle trafficking by allowing better understanding of different synaptic vesicle pools within the nerve terminal, vesicle intermixing, and vesicle clustering at release sites. Finally, the dyes are now being used in innovative ways to reveal further insights into synaptic plasticity.     

Bassoon and the synaptic ribbon organize Ca²+ channels and vesicles to add release sites and promote refilling

At the presynaptic active zone, Ca2+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca2+ channel clustering and synaptic vesicle docking are organized. Here, we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses--mostly lacking the ribbon--showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca2+ channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca2+ channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis was reduced, notwithstanding normal coupling of the remaining Ca2+ channels to exocytosis. In vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in vivo data independently supported morphological and functional in vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca2+ channels and vesicles, and (2) promote vesicle replenishment.     

The neurotransmitter cycle and quantal size

Changes in the response to release of a single synaptic vesicle have generally been attributed to postsynaptic modification of receptor sensitivity, but considerable evidence now demonstrates that alterations in vesicle filling also contribute to changes in quantal size. Receptors are not saturated at many synapses, and changes in the amount of transmitter per vesicle contribute to the physiological regulation of release. On the other hand, the presynaptic factors that determine quantal size remain poorly understood. Aside from regulation of the fusion pore, these mechanisms fall into two general categories: those that affect the accumulation of transmitter inside a vesicle and those that affect vesicle size. This review will summarize current understanding of the neurotransmitter cycle and indicate basic, unanswered questions about the presynaptic regulation of quantal size.     

Presynaptic regulation of neurotransmission in Drosophila by the g protein-coupled receptor methuselah

Regulation of synaptic strength is essential for neuronal information processing, but the molecular mechanisms that control changes in neuroexocytosis are only partially known. Here we show that the putative G protein-coupled receptor Methuselah (Mth) is required in the presynaptic motor neuron to acutely upregulate neurotransmitter exocytosis at larval Drosophila NMJs. Mutations in the mth gene reduce evoked neurotransmitter release by approximately 50%, and decrease synaptic area and the density of docked and clustered vesicles. Pre- but not postsynaptic expression of normal Mth restored normal release in mth mutants. Conditional expression of Mth restored normal release and normal vesicle docking and clustering but not the reduced size of synaptic sites, suggesting that Mth acutely adjusts vesicle trafficking to synaptic sites.     

Calcium influx and mitochondrial alterations at synapses exposed to snake neurotoxins or their phospholipid hydrolysis products

Snake presynaptic phospholipase A2 neurotoxins (SPANs) bind to the presynaptic membrane and hydrolyze phosphatidylcholine with generation of lysophosphatidylcholine (LysoPC) and fatty acid (FA). The LysoPC+FA mixture promotes membrane fusion, inducing the exocytosis of the ready-to-release synaptic vesicles. However, also the reserve pool of synaptic vesicles disappears from nerve terminals intoxicated with SPAN or LysoPC+FA. Here, we show that LysoPC+FA and SPANs cause a large influx of extracellular calcium into swollen nerve terminals, which accounts for the extensive synaptic vesicle release. This is paralleled by the change of morphology and the collapse of membrane potential of mitochondria within nerve bulges. These results complete the picture of events occurring at nerve terminals intoxicated by SPANs and define the LysoPC+FA lipid mixture as a novel and effective agonist of synaptic vesicle release.