Synaptic vesicles: half full or half empty?

The regulation of quantal size through pre- rather than postsynaptic mechanisms has recently received considerable attention as a potential mechanism for plasticity. Vesicular transporters catalyze the filling of synaptic vesicles with transmitter and are thus potential substrates for such presynaptic regulation. In this issue of Neuron, Prado et al. pursue this line of investigation and show that changes in transporter expression that alter quantal size can affect behavior.     

Presynaptic control of quantal size: kinetic mechanisms and implications for synaptic transmission and plasticity

Although the strength of quantal synaptic transmission is jointly controlled by pre- and post-synaptic mechanisms, the presynaptic mechanisms remain substantially less well characterized. Recent studies reveal that a single package of neurotransmitter is generally insufficient to activate all available postsynaptic receptors, whereas the sum of transmitter from multiple vesicles can result in receptor saturation. Thus, depending upon the number of vesicles released, a given synaptic pathway might be either 'reliable' or 'unreliable'. A lack of receptor saturation in turn makes it possible to modify quantal size by altering the flux of transmitter through the synaptic cleft. Studies are now illuminating several new mechanisms behind the regulation of this transmitter flux--characteristics that control how transmitter is loaded into vesicles, how it is released and the manner by which it interacts with postsynaptic receptors.     

Presynaptic Control of Glycine Transporter 2 (GlyT2) by Physical and Functional Association with Plasma Membrane Ca2+-ATPase (PMCA) and Na+-Ca2+ Exchanger (NCX)

Fast inhibitory glycinergic transmission occurs in spinal cord, brainstem, and retina to modulate the processing of motor and sensory information. After synaptic vesicle fusion, glycine is recovered back to the presynaptic terminal by the neuronal glycine transporter 2 (GlyT2) to maintain quantal glycine content in synaptic vesicles. The loss of presynaptic GlyT2 drastically impairs the refilling of glycinergic synaptic vesicles and severely disrupts neurotransmission. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans. Here, we show a novel endogenous regulatory mechanism that can modulate GlyT2 activity based on a compartmentalized interaction between GlyT2, neuronal plasma membrane Ca²⁺-ATPase (PMCA) isoforms 2 and 3, and Na⁺/Ca²⁺-exchanger 1 (NCX1). This GlyT2·PMCA2,3·NCX1 complex is found in lipid raft subdomains where GlyT2 has been previously found to be fully active. We show that endogenous PMCA and NCX activities are necessary for GlyT2 activity and that this modulation depends on lipid raft integrity. Besides, we propose a model in which GlyT2·PMCA2–3·NCX complex would help Na⁺/K⁺-ATPase in controlling local Na⁺ increases derived from GlyT2 activity after neurotransmitter release.     

Inhibition of vesicular glutamate storage and exocytotic release by Rose Bengal

It had been thought that quantal size in synaptic transmission is invariable. Evidence has been emerging, however, that quantal size can be varied under certain conditions. We present evidence that alteration in vesicular [(3)H]L-glutamate (Glu) content within the synaptosome (a pinched-off nerve ending preparation) leads to a change in the amount of exocytotically released [(3)H]Glu. We found that Rose Bengal, a polyhalogenated fluorescein derivative, is a quite potent membrane-permeant inhibitor (K(i) = 19 nM) of glutamate uptake into isolated synaptic vesicles. This vesicular Glu uptake inhibition was achieved largely without affecting H(+)-pump ATPase. We show that various degrees of reduction elicited by Rose Bengal in [(3)H]Glu in synaptic vesicles inside the synaptosome result in a corresponding decrease in the amount of [(3)H]Glu released in a depolarization- (induced by 4-aminopyridine) and Ca(2+)-dependent manner. In contrast, fluorescein, the halogen-free analog of Rose Bengal, which is devoid of inhibitory activity on vesicular [(3)H]Glu uptake, failed to change the amount of exocytotically released [(3)H]Glu. These observations suggest that glutamate synaptic transmission could be altered by pharmacological intervention of glutamate uptake into synaptic vesicles in the nerve terminal, a new mode of synaptic manipulation for glutamate transmission.     

Activity-dependent remodeling of presynaptic inputs by postsynaptic expression of activated CaMKII

Competitive synaptic remodeling is an important feature of developmental plasticity, but the molecular mechanisms remain largely unknown. Calcium/calmodulin-dependent protein kinase II (CaMKII) can induce postsynaptic changes in synaptic strength. We show that postsynaptic CaMKII also generates structural synaptic rearrangements between cultured cortical neurons. Postsynaptic expression of activated CaMKII (T286D) increased the strength of transmission between pairs of pyramidal neuron by a factor of 4, through a modest increase in quantal amplitude and a larger increase in the number of synaptic contacts. Concurrently, T286D reduced overall excitatory synaptic density and increased the proportion of unconnected pairs. This suggests that connectivity from some synaptic partners was increased while other partners were eliminated. The enhancement of connectivity required activity and NMDA receptor activation, while the elimination did not. These data suggest that postsynaptic activation of CaMKII induces a structural remodeling of presynaptic inputs that favors the retention of active presynaptic partners.     

Regulation of synaptic vesicles pools within motor nerve terminals during short-term facilitation and neuromodulation.

The reserve pool (RP) and readily releasable pool (RRP) of synaptic vesicles within presynaptic nerve terminals were physiologically differentiated into distinctly separate functional groups. This was accomplished in glutamatergic nerve terminals by blocking the glutamate transporter with dl-threo-beta-benzyloxyaspartate (TBOA; 10 microM) during electrical stimulation with either 40 Hz of 10 pulses within a train or 20- or 50-Hz continuous stimulation. The 50-Hz continuous stimulation decreased the excitatory postsynaptic potential amplitude 60 min faster than for the 20-Hz continuous stimulation in the presence of TBOA (P < 0.05). There was no significant difference between the train stimulation and 20-Hz continuous stimulation in the run-down time in the presence of TBOA. After TBOA-induced synaptic depression, the excitatory postsynaptic potentials were rapidly (<1 min) revitalized by exposure to serotonin (5-HT, 1 microM) in every preparation tested (P < 0.05). At this glutamatergic nerve terminal, 5-HT promotes an increase probability of vesicular docking and fusion. Quantal recordings made directly at nerve terminals revealed smaller quantal sizes with TBOA exposure with a marked increase in quantal size as well as a continual appearance of smaller quanta upon 5-HT treatment after TBOA-induced depression. Thus 5-HT was able to recruit vesicles from the RP that were not rapidly depleted by acute TBOA treatment and electrical stimulation. The results support the notion that the RRP is selectively activated during rapid electrical stimulation sparing the RP; however, the RP can be recruited by the neuromodulator 5-HT. This suggests at least two separate kinetic and distinct regulatory paths for vesicle recycling within the presynaptic nerve terminal.     

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.     

Reduced SNAP‐25 alters short‐term plasticity at developing glutamatergic synapses

SNAP‐25 is a key component of the synaptic‐vesicle fusion machinery, involved in several psychiatric diseases including schizophrenia and ADHD. SNAP‐25 protein expression is lower in different brain areas of schizophrenic patients and in ADHD mouse models. How the reduced expression of SNAP‐25 alters the properties of synaptic transmission, leading to a pathological phenotype, is unknown. We show that, unexpectedly, halved SNAP‐25 levels at 13–14 DIV not only fail to impair synaptic transmission but instead enhance evoked glutamatergic neurotransmission. This effect is possibly dependent on presynaptic voltage‐gated calcium channel activity and is not accompanied by changes in spontaneous quantal events or in the pool of readily releasable synaptic vesicles. Notably, synapses of 13–14 DIV neurons with reduced SNAP‐25 expression show paired‐pulse depression as opposed to paired‐pulse facilitation occurring in their wild‐type counterparts. This phenotype disappears with synapse maturation. As alterations in short‐term plasticity represent a new mechanism contributing to cognitive impairments in intellectual disabilities, our data provide mechanistic clues for neuronal circuit alterations in psychiatric diseases characterized by reduced expression of SNAP‐25.     

Quantal analysis of synaptic processes in the neocortex

The application of fluctuation analysis to studies of synaptic function in the neocortex is discussed. Analysis of failures of transmission has been valuable in indicating whether a presynaptic or a postsynaptic site is responsible for a change in synaptic efficacy. When combined with detailed ultrastructural verification of all synapses involved in an individual cell to cell connection, a reasonable estimate of quantal size and release probability under conditions of low frequency activity can be obtained. However, both the number of available release sites in functional terms and the probability that an action potential (AP) will release transmitter from any given site can vary from AP to AP at higher frequencies. A variety of presynaptic mechanisms that modulate release are now apparent. For example, one mechanism dominates release patterns at one class of connection which is insensitive to absolute firing frequency, but responsive to changes in frequency. At another class of connection, a different mechanism dominates, resulting in high sensitivity to frequency.     

神经元突触前可塑性的结构及分子基础

突触可塑性是神经元间信息传递的重要生理调控机制,它包括突触前可塑性和突触后可塑性.突触前可塑性是指通过对神经递质释放过程的干预、修饰,调节突触强度的过程.突触强度的变化,是通过影响量子的大小,活动区的个数和囊泡释放概率来实现的.而突触前囊泡活动尤为重要：从转运、搭靠、融合至内吞进入下一轮循环,每一步都是由一群互相作用的蛋白质共同完成的.     

Calmodulin mediates rapid recruitment of fast-releasing synaptic vesicles at a calyx-type synapse.

In many synapses, depletion and recruitment of releasable synaptic vesicles contribute to use-dependent synaptic depression and recovery. Recently it has been shown that high-frequency presynaptic stimulation enhances recovery from depression, which may be mediated by Ca2+. We addressed this issue by measuring quantal release rates at the calyx of Held synapse and found that transmission is mediated by a heterogeneous population of vesicles, with one subset releasing rapidly and recovering slowly and another one releasing reluctantly and recovering rapidly. Ca2+ promotes refilling of the rapidly releasing synaptic vesicle pool and calmodulin inhibitors block this effect. We propose that calmodulin-dependent refilling supports recovery from synaptic depression during high-frequency trains in concert with rapid recovery of the slowly releasing vesicles.     

Building a bilayer model of the neuromuscular synapse

Progress over the past 10 years has made it possible to construct a simple model of neurotransmitter release. Currently, some models use artificially formed vesicles to represent synaptic vesicles and a planar lipid bilayer as a presynaptic membrane. Fusion of vesicles with the bilayer is via channel proteins in the vesicle membrane and an osmotic gradient. In this paper, a framework is presented for the successful construction of a more complete model of synaptic transmission. This model includes real synaptic vesicles that fuse with a planar bilayer. The bilayer contains acetylcholine receptor (AChR) channels which function as autoreceptors in the membrane. Vesicle fusion is initiated following a Ca²⁺ flux through voltage-gated Ca²⁺ channels. Key steps in the plan are validated by mathematical modeling. Specifically, the probability that a reconstituted AChR channel opens following the release of ACh from a fusing vesicle, is calculated as a function of time, quantal content, and number of reconstituted AChRs. Experimentally obtainable parameters for construction of a working synapse are given. The inevitable construction of a full working model will mean that the minimal structures necessary for synaptic transmission are identified. This will open the door in determining regulatory and modulatory factors of transmitter release.     

ELECTRON MICROSCOPY OF SYNAPTIC STRUCTURE OF OCTOPUS BRAIN

The well known type of synapse between a presynaptic process containing vesicles and a "clear" postsynaptic process can be commonly observed in the various lobes of the brain of Octopus. The presynaptic vesicles are aggregated near regions of the synaptic membranes which show specialisation and asymmetric "thickening" indicating functional polarisation, and here chemical transmission is presumed to take place. In addition, in the vertical lobe a very interesting serial arrangement of synaptic contacts occurs. Presynaptic bags, formed from varicosities of fibres from the superior frontal lobe, contact the trunks of amacrine cells in the manner just described. The trunks, however, although apparently postsynaptic are themselves packed with synaptic vesicles. The trunks, in turn, make "presynaptic" contacts with clear spinous processes of other neurons of yet undetermined origin. Typical polarised membrane specialisations occur at the contact regions. The trunk vesicles aggregated closest to the contact regions have a shell of particles round their walls. At present, there is no way of telling whether the membrane conductance to the various ions is differently affected at either of the transmission sites, and, if an inhibitory mechanism is involved, whether it is of the presynaptic or postsynaptic variety.     

Bassoon speeds vesicle reloading at a central excitatory synapse

Sustained rate-coded signals encode many types of sensory modalities. Some sensory synapses possess specialized ribbon structures, which tether vesicles, to enable high-frequency signaling. However, central synapses lack these structures, yet some can maintain signaling over a wide bandwidth. To analyze the underlying molecular mechanisms, we investigated the function of the active zone core component Bassoon in cerebellar mossy fiber to granule cell synapses. We show that short-term synaptic depression is enhanced in Bassoon knockout mice during sustained high-frequency trains but basal synaptic transmission is unaffected. Fluctuation and quantal analysis as well as quantification with constrained short-term plasticity models revealed that the vesicle reloading rate was halved in the absence of Bassoon. Thus, our data show that the cytomatrix protein Bassoon speeds the reloading of vesicles to release sites at a central excitatory synapse.     

Effective Mechanism for Synthesis of Neurotransmitter Glutamate and its Loading into Synaptic Vesicles

Glutamate accumulation into synaptic vesicles is a pivotal step in glutamate transmission. This process is achieved by a vesicular glutamate transporter (VGLUT) coupled to v-type proton ATPase. Normal synaptic transmission, in particular during intensive neuronal firing, would demand rapid transmitter re-filling of emptied synaptic vesicles. We have previously shown that isolated synaptic vesicles are capable of synthesizing glutamate from α-ketoglutarate (not from glutamine) by vesicle-bound aspartate aminotransferase for immediate uptake, in addition to ATP required for uptake by vesicle-bound glycolytic enzymes. This suggests that local synthesis of these substances, essential for glutamate transmission, could occur at the synaptic vesicle. Here we provide evidence that synaptosomes (pinched-off nerve terminals) also accumulate α-ketoglutarate-derived glutamate into synaptic vesicles within, at the expense of ATP generated through glycolysis. Glutamine-derived glutamate is also accumulated into synaptic vesicles in synaptosomes. The underlying mechanism is discussed. It is suggested that local synthesis of both glutamate and ATP at the presynaptic synaptic vesicle would represent an efficient mechanism for swift glutamate loading into synaptic vesicles, supporting maintenance of normal synaptic transmission.     

Localization effects of acetylcholine release from a synaptic vesicle at the neuromuscular junction.

A two-dimensional compartment model devised for the appropriate representation of the transient process of the spontaneous generation of miniature endplate current (MEPC) at the neuromuscular junction is applied for clarifying the biochemical significance of the quantal release mechanism of acetylcholine (ACh), a typical neurotransmitter, in the synaptic chemical transmission process. The simulation analysis with the model demonstrates that the localization of the ACh release due to the fusion of a synaptic vesicle with the presynaptic membrane has significant effects on the amplitude of MEPC and that the stronger effects are caused with the smaller diffusion coefficients of ACh in the cleft. The sharpest and highest response of MEPC is achieved when the release area is about 4 times to the natural release through the narrow pore. On the other hand, the actual localization corresponding to the natural release of ACh makes the amplitude of MEPC higher by a factor about 2.5 compared with that in the most extended release of ACh examined, implying that the natural release mechanism works as an amplifier of the MEPC with the fixed amount of ACh available.     

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.     

Do mental events cause neural events analogously to the probability fields of quantum mechanics?

If non-material mental events, such as the intention to carry out an action, are to have an effective action on neural events in the brain, it has to be at the most subtle and plastic level of these events. In the first stage of our enquiry an introduction to conventional synaptic theory leads on to an account of the manner of operation of the ultimate synaptic units. These units are the synaptic boutons that, when excited by an all-or-nothing nerve impulse, deliver the total contents of a single synaptic vesicle, not regularly, but probabilistically. This quantal emission of the synaptic transmitter molecules (about 5000-10 000) is the elementary unit of the transmission process from one neuron to another. In the second stage this refined physiological analysis leads on to an account of the ultrastructure of the synapse, which gives clues as to the manner of its unitary probabilistic operation. The essential feature is that the effective structure of each bouton is a paracrystalline presynaptic vesicular grid with about 50 vesicles, which acts probabilistically in vesicular (quantal) release. In the third stage it is considered how a non-material mental event, such as an intention to move, could influence the subtle probabilistic operations of synaptic boutons. On the biological side, attention is focused on the paracrystalline presynaptic vesicular grids as the targets for non-material mental events. On the physical side, attention is focused on the probabilistic fields of quantum mechanics which carry neither mass nor energy, but which nevertheless can exert effective action at microsites. The new light on the mind-brain problem came from the hypothesis that the non-material mental events, the 'World 2' of Popper, relate to the neural events of the brain (the 'World 1' of matter and energy) by actions in conformity with quantum theory. This hypothesis that mental events act on probabilistic synaptic events in a manner analogous to the probability fields of quantum mechanics seems to open up an immense field of scientific investigation both in quantum physics and in neuroscience.     

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.