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.     

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.     

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.     

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.     

Regulation of synaptic vesicle docking by different classes of macromolecules in active zone material

The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10-15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles' distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry.     

Quantal release of serotonin

We have studied the origin of quantal variability for small synaptic vesicles (SSVs) and large dense-cored vesicles (LDCVs). As a model, we used serotonergic Retzius neurons of leech that allow for combined amperometrical and morphological analyses of quantal transmitter release. We find that the transmitter amount released by a SSV varies proportionally to the volume of the vesicle, suggesting that serotonin is stored at a constant intravesicular concentration and is completely discharged during exocytosis. Transmitter discharge from LDCVs shows a higher degree of variability than is expected from their size distribution, and bulk release from LDCVs is slower than release from SSVs. On average, differences in the transmitter amount released from SSVs and LDCVs are proportional to the size differences of the organelles, suggesting that transmitter is stored at similar concentrations in SSVs and LDCVs.     

A single packet of transmitter does not saturate postsynaptic glutamate receptors

Neurotransmitter is stored in synaptic vesicles and released by exocytosis into the synaptic cleft. One of the fundamental questions in central synaptic transmission is whether a quantal packet of transmitter saturates postsynaptic receptors. To address this question, we loaded the excitatory transmitter L-glutamate via whole-cell recording pipettes into the giant nerve terminal, the calyx of Held, in rat brainstem slices. This caused marked potentiations of both quantal and action potential-evoked EPSCs mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors. These results directly demonstrate that neither AMPA nor NMDA receptors are saturated by a single packet of transmitter, and indicate that vesicular transmitter content is an important determinant of synaptic efficacy.     

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.     

Structural changes after transmitter release at the frog neuromuscular junction

The sequence of structural changes that occur during synaptic vesicle exocytosis was studied by quick-freezing muscles at different intervals after stimulating their nerves, in the presence of 4-aminopyridine to increase the number of transmitter quanta released by each stimulus. Vesicle openings began to appear at the active zones of the intramuscular nerves within 3-4 ms after a single stimulus. The concentration of these openings peaked at 5-6 ms, and then declined to zero 50-100 ms late. At the later times, vesicle openings tended to be larger. Left behind at the active zones, after the vesicle openings disappeared, were clusters of large intramembrane particles. The larger particles in these clusters were the same size as intramembrane particles in undischarged vesicles, and were slightly larger than the particles which form the rows delineating active zones. Because previous tracer work had shown that new vesicles do not pinch off from the plasma membrane at these early times, we concluded that the particle clusters originate from membranes of discharged vesicles which collapse into the plasmalemma after exocytosis. The rate of vesicle collapse appeared to be variable because different stages occurred simultaneously at most times after stimulation; this asynchrony was taken to indicate that the collapse of each exocytotic vesicle is slowed by previous nearby collapses. The ultimate fate of synaptic vesicle membrane after collapse appeared to be coalescence with the plasma membrane, as the clusters of particles gradually dispersed into surrounding areas during the first second after a stimulus. The membrane retrieval and recycling that reverse this exocytotic sequence have a slower onset, as has been described in previous reports.     

The molecular mechanism of nondegranulative release of biogenic amines.

According to current teaching biogenic amines are released by exocytosis, i.e. by evacuation of amine storing vesicles or granules into the extracellular space. The release of transmitter amines is quantal, i.e. occurs in packs of transmitter molecules. These packs are assumed to be identical with vesicle contents, in other words, the smallest releasable quantum equals the amine content of one vesicle. However, there are experimental observations which do not fit in with this version of an exocytotic release theory. Observed quantitative discrepancies could be explained if the release mechanism allowed a fractional release of transmitter amine from several vesicles instead of the total evacuation of a few. The lack of adequate knowledge about the mechanisms of storage of biogenic amines within the vesicles has up til now rendered it difficult to envisage the machinery behind a fractional release of the amine content of a vesicle. In extensive in-vitro studies we have found that the matrices of amine storing granules (i.e. from mast cells, chromaffin cells and nerve terminals) show the properties of weak cation exchanger materials, carboxyl groups serving as amine binding ionic sites. When exposed to cations like sodium and potassium ions, the amines are released from their storage sites according to kinetics characteristic of weak cation exchangers. In vivo, amine release from cat adrenals on splanchnic nerve stimulation also occurs according to ion exchange kinetics. Histamine release from mast cells is considered to occur as the result of degranulation, i.e. the expulsion of histamine storing granules to the extracellular space, a typical example of exocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The kinetics of nerve-evoked quantal secretion

Current views on quantal release of neurotransmitters hold that after the vesicle migrates towards release sites (active zones), multiple protein interactions mediate the docking of the vesicle to the presynaptic membrane and the formation of a multimolecular protein complex (the 'fusion machine') which ultimately makes the vesicle competent to release a quantum in response to the action potential. Classical biophysical studies of quantal release have modelled the process by a binomial system where n vesicles (sites) competent for exocytosis release a quantum, with probability p, in response to the action potential. This is likely to be an oversimplified model. Furthermore, statistical and kinetic studies have given results which are difficult to reconcile within this framework. Here, data are presented and discussed which suggest a revision of the biophysical model. Transient silencing of release is shown to occur following the pulse of synchronous transmitter release, which is evoked by the presynaptic action potential. This points to a schema where the vesicle fusion complex assembly is a reversible, stochastic process. Asynchronous exocytosis may occur at several intermediate stages in the process, along paths which may be differentially regulated by divalent cations or other factors. The fusion complex becomes competent for synchronous release (armed vesicles) only at appropriately organized sites. The action potential then triggers (deterministically rather than stochastically) the synchronous discharge of all armed vesicles. The existence of a specific conformation for the fusion complex to be competent for synchronous evoked fusion reconciles statistical and kinetic results during repetitive stimulation and helps explain the specific effects of toxins and genetic manipulation on the synchronization of release in response to an action potential.     

Vesicular Monogamy?

Vesicular neurotransmitter transporters package transmitter into the lumen of synaptic vesicles for quantal release. However, the number of transporters that localize to each vesicle is not known. In this issue of Neuron, a study by Daniels et al. using the Drosophila neuromuscular junction and mutations of the vesicular glutamate transporter suggests that one transporter may suffice to fill each vesicle.     

Vesicular and Plasma Membrane Transporters for Neurotransmitters

The regulated exocytosis that mediates chemical signaling at synapses requires mechanisms to coordinate the immediate response to stimulation with the recycling needed to sustain release. Two general classes of transporter contribute to release, one located on synaptic vesicles that loads them with transmitter, and a second at the plasma membrane that both terminates signaling and serves to recycle transmitter for subsequent rounds of release. Originally identified as the target of psychoactive drugs, these transport systems have important roles in transmitter release, but we are only beginning to understand their contribution to synaptic transmission, plasticity, behavior, and disease. Recent work has started to provide a structural basis for their activity, to characterize their trafficking and potential for regulation. The results indicate that far from the passive target of psychoactive drugs, neurotransmitter transporters undergo regulation that contributes to synaptic plasticity.The speed and potency of synaptic transmission depend on the immediate availability of synaptic vesicles filled with high concentrations of neurotransmitter. In this article, we focus on the mechanisms responsible for packaging transmitter into synaptic vesicles and for reuptake from the extracellular space that both terminates synaptic transmission and recycles transmitter for future rounds of release. Collectively, we refer to this entire process as the neurotransmitter cycle.The recycling of neurotransmitter illustrates a general, conceptual problem for the mechanism of vesicular release. At the plasma membrane, more active reuptake should help to replenish the pool of releasable transmitter, but may also reduce the extent and duration of signaling to the postsynaptic cell. Conversely, loss of reuptake increases the activation of receptors but results in the depletion of stores (Jones et al. 1998). At the vesicle, steeper concentration gradients release more transmitter per vesicle but reduce the cytosolic transmitter available for refilling, whereas more shallow gradients facilitate refilling but reduce the transmitter available for release. The way in which the nerve terminal balances these competing factors thus has profound consequences for synaptic transmission.     

Quantal size is dependent on stimulation frequency and calcium entry in calf chromaffin cells

To what extent the quantal hypothesis of transmitter release applies to dense-core vesicle (DCV) secretion is unknown. We determined the characteristics of individual secretory events in calf chromaffin cells using catecholamine amperometry combined with different patterns of stimulation. Raising the frequency of action potential trains from 0.25-10 Hz in 2 mM [Ca(2+)]o or [Ca(2+)]o from 0.25-7 mM at 7 Hz elevated the amount released per event (quantal size). With increased stimulation, quantal size rose continuously, not abruptly, suggesting that release efficiency from a single population of DCVs rather than recruitment of different-sized vesicles contributed to the effect. These results suggest that catecholamine secretion does not conform to the quantal model. Inhibition of rapid endocytosis damped secretion in successive episodes, implying an essential role for this process in the recycling of vesicles needed for continuous secretion.     

The probability of quantal secretion within an array of calcium channels of an active zone

A Monte Carlo analysis has been made of calcium dynamics in submembranous domains of active zones in which the calcium contributed by the opening of many channels is pooled. The kinetics of calcium ions in these domains has been determined using simulations for channels arranged in different geometries, according to the active zone under consideration: rectangular grids for varicosities and boutons and lines for motor-nerve terminals. The effects of endogenous fixed and mobile buffers on the two-dimensional distribution of free calcium ions at these active zones are then given, together with the extent to which these are perturbed and can be detected with different affinity calcium indicators when the calcium channels open stochastically under an action potential. A Monte Carlo analysis of how the dynamics of calcium ions in the submembranous domains determines the probability of exocytosis from docked vesicles is also presented. The spatial distribution of exocytosis from rectangular arrays of secretory units is such that exocytosis is largely excluded from the edges of the array, due to the effects of endogenous buffers. There is a steeper than linear increase in quantal release with an increase in the number of secretory units in the array, indicating that there is not just a local interaction between secretory units. Conditioning action potentials promote an increase in quantal release by a subsequent action potential primarily by depleting the fixed and mobile buffers in the center of the array. In the case of two parallel lines of secretory units exocytosis is random, and diffusion, together with the endogenous calcium buffers, ensures that the secretory units only interact over relatively short distances. As a consequence of this and in contrast to the case of the rectangular array, there is a linear relationship between the extent of quantal secretion from these zones and their length, for lengths greater than a critical value. This Monte Carlo analysis successfully predicts the relationship between the size and geometry of active zones and the probability of quantal secretion at these, the existence of quantal versus multiquantal release at different active zones, and the origins of the F1 phase of facilitation in synapses possessing different active zone geometries.     

A model for exocytosis based on the opening of calcium-activated potassium channels in vesicles

It is proposed that the role of calcium in calcium-induced exocytosis is to open Ca-activated K channels present in vesicle membranes. The opening of these channels coupled with anion transport across the vesicle membranes would result in an influx of K and anions, increasing the osmotic pressure of the vesicles. For those vesicles situated very close to the cell plasma membrane, this would lead to fusion with the membrane and exocytosis of the vesicle contents. This model can account for facilitation and other key properties of transmitter release. In addition, the model predicts that vesicles with a higher transmitter content, and hence higher initial osmotic pressure, would be preferentially discharged. The model also predicts that a faster response can be obtained for small vesicles than for large vesicles, providing a rationale as to why neurotransmitters, which must be released quickly, are packaged in small vesicles.     

Complexin arrests a pool of docked vesicles for fast Ca(2+)-dependent release

Regulated exocytosis requires that the assembly of the basic membrane fusion machinery is temporarily arrested. Synchronized membrane fusion is then caused by a specific trigger-a local rise of the Ca(2+) concentration. Using reconstituted giant unilamellar vesicles (GUVs), we have analysed the role of complexin and membrane-anchored synaptotagmin 1 in arresting and synchronizing fusion by lipid-mixing and cryo-electron microscopy. We find that they mediate the formation and consumption of docked small unilamellar vesicles (SUVs) via the following sequence of events: Synaptotagmin 1 mediates v-SNARE-SUV docking to t-SNARE-GUVs in a Ca(2+)-independent manner. Complexin blocks vesicle consumption, causing accumulation of docked vesicles. Together with synaptotagmin 1, complexin synchronizes and stimulates rapid fusion of accumulated docked vesicles in response to physiological Ca(2+) concentrations. Thus, the reconstituted assay resolves both the stimulatory and inhibitory function of complexin and mimics key aspects of synaptic vesicle fusion.     

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.     

Synaptic vesicles immunoisolated from rat cerebral cortex contain high levels of glutamate

L-Glutamate is regarded as the major excitatory neurotransmitter in the mammalian CNS. However, whether the released transmitter originates from a cytosolic pool or is discharged from synaptic vesicles by exocytosis (vesicle hypothesis) remains controversial. A problem with the general acceptance of the vesicle hypothesis is that the enrichment of glutamate in synaptic vesicles has not been convincingly demonstrated. In the present study, we have analyzed the glutamate content of synaptic vesicles isolated from rat cerebral cortex by a novel immunobead procedure. A large amount of glutamate was present in these vesicles when a proton electrochemical gradient was maintained across the vesicle membrane during isolation. Compared with the starting fraction, glutamate was enriched more than 10-fold relative to other amino acids. Addition of N-ethylmaleimide prevented glutamate loss during isolation. Isotope exchange experiments revealed that exchange or re-uptake of glutamate after homogenization is negligible. We conclude that rat brain synaptic vesicles contain high levels of glutamate in situ.     

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.