Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire

Two vesicle pools, readily releasable (RRP) and reserve (RP) pools, are present at Drosophila neuromuscular junctions. Using a temperature-sensitive mutant, shibire(ts), we studied pool sizes and vesicle mobilization rates. In shibire(ts), due to lack of endocytosis at nonpermissive temperatures, synaptic currents continuously declined during tetanic stimulation until they ceased as the result of vesicle depletion. By then, approximately 84,000 quanta were released. Vesicles were mobilized from RP at a rate 1/7-1/10 of RRP. Cytochalasin D inhibited mobilization of vesicles from RP, allowing us to estimate the size of RRP as 14%-19% of all vesicles. Vesicle recycling supports synaptic transmission during prolonged tetanic stimulation and the maximum recycling rate was 1000 vesicles/s.     

Synaptic vesicle pools at the frog neuromuscular junction

We have characterized the morphological and functional properties of the readily releasable pool (RRP) and the reserve pool of synaptic vesicles in frog motor nerve terminals using fluorescence microscopy, electron microscopy, and electrophysiology. At rest, about 20% of vesicles reside in the RRP, which is depleted in about 10 s by high-frequency nerve stimulation (30 Hz); the RRP refills in about 1 min, and surprisingly, refilling occurs almost entirely by recycling, not mobilization from the reserve pool. The reserve pool is depleted during 30 Hz stimulation with a time constant of about 40 s, and it refills slowly (half-time about 8 min) as nascent vesicles bud from randomly distributed cisternae and surface membrane infoldings and enter vesicle clusters spaced at regular intervals along the terminal. Transmitter output during low-frequency stimulation (2-5 Hz) is maintained entirely by RRP recycling; few if any vesicles are mobilized from the reserve pool.     

The effects of hydrogen sulfide on the processes of exo- and endocytosis of synaptic vesicles in the mouse motor nerve endings

The effects of sodium hydrosulfide (NaHS), the donor of hydrogen sulfide (H2S), on the exo/endocytosis cycle of synaptic vesicles in the motor nerve ending of the mouse diaphragm were studied using intracellular microelectrode technique and fluorescent microscopy. NaHS increased the frequency of miniature end-plate potentials (MEPPs), without changing their amplitude-time parameters. NaHS also increased the amplitude of the evoked postsynaptic responses during single stimulation (0.3 Hz), which was the evidence of the enhanced synaptic vesicle exocytosis. During high-frequency stimulation (50 Hz), NaHS induced more significant decline of neurotransmitter release, probably due to the lower rate of synaptic vesicle mobilization from recycling pool to exocytic sites. NaHS also decreased the uptake of the fluorescent endocytic dye FM 1–43, which indicated the reduced endocytosis of synaptic vesicles. Thus, the H₂S donor increases exocytosis and decreases the processes of synaptic vesicle endocytosis and mobilization in the mouse motor nerve ending.     

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.     

Inhibition of exocytosis or endocytosis blocks activity-dependent redistribution of synapsin

The synaptic vesicle cycle encompasses the pre-synaptic events that drive neurotransmission. Influx of calcium leads to the fusion of synaptic vesicles with the plasma membrane and the release of neurotransmitter, closely followed by endocytosis. Vacated release sites are repopulated with vesicles which are then primed for release. When activity is intense, reserve vesicles may be mobilized to counteract an eventual decline in transmission. Recently, interplay between endocytosis and repopulation of the readily releasable pool of vesicles has been identified. In this study, we show that exo-endocytosis is necessary to enable detachment of synapsin from reserve pool vesicles during synaptic activity. We report that blockage of exocytosis in cultured mouse hippocampal neurons, either by tetanus toxin or by the deletion of munc13, inhibits the activity-dependent redistribution of synapsin from the pre-synaptic terminal into the axon. Likewise, perturbation of endocytosis with dynasore or by a dynamin dominant-negative mutant fully prevents synapsin redistribution. Such inhibition of synapsin redistribution occurred despite the efficient phosphorylation of synapsin at its protein kinase A/CaMKI site, indicating that disengagement of synapsin from the vesicles requires exocytosis and endocytosis in addition to phosphorylation. Our results therefore reveal hitherto unidentified feedback within the synaptic vesicle cycle involving the synapsin-managed reserve pool.     

Peculiarities of synaptic vesicle recycling in frog and mouse motor nerve terminals

Using electrophysiology and fluorescence microscopy with dye FM 1-43, a comparative study of peculiarities of neurotransmitter secretion, synaptic vesicle exo-endocytosis and recycling has been carried out in nerve terminals (NT) of the skin-sternal muscle of the frog Rana ridibunda and of the white mouse diaphragm muscle during a long-term high-frequency stimulation (20 imp/s). The obtained data have allowed identifying three synaptic vesicle pools and two recycling ways in the motor NT. In the frog NT, the long-term high-frequency stimulation induced consecutive expenditure of the pool ready to release, the mobilizational, and reserve vesicle pools. The exocytosis rate exceeded markedly the endocytosis rate; the slow synaptic vesicle recycling with replenishment of the reserve pool was predominant. In the mouse NT, only the vesicles of the ready to release and the mobilizational pools, which are replenished predominantly by fast recycling, were exocytosed. The exo- and endocytosis occurred practically in parallel, while vesicles of the reserve pool did not participate in the neurotransmitter secretion. It is suggested that evolution of the motor NT from the poikilothermal to homoiothermal animals went by the way of a decrease of the vesicle pool size, the more economic expenditure and the more effective reuse of synaptic vesicles owing to the high rates of endocytosis and recycling. These peculiarities can provide in NT of homoiothermal animals a long maintenance of neurotransmitter secretion at the steady and sufficiently high level to preserve reliability of synaptic transmission in the process of the high-frequency activity.     

Vesicle cycle in mouse diaphragm motor nerve terminals

In our research on mouse diaphragm muscles the dynamic of neurotransmitter secretion and synaptic vesicles recycling (exo-endocytosis cycle) at the long-term rhythmic stimulation (20Hz) are explored using an intracellular microelectrode registration and a fluorescent microscopy. It have been shown, thate change of end plant potentials (EPP) amplitude at the rhythmic training occurs in three phases: initial transient decrease, long amplitude stabilization (1-2 min)--the plateau and secondary slow decrease. After 3 minute stimulations the EPP amplitude recovery observed during several seconds. Loading the synaptic vesicle by fluorescent endocytic dye FM 1-43 had shown that the rhythmic stimulation results to gradual (during 5-6 mines) fluorescence decrease in NT, indicating the synaptic vesicle exocytosis. The quantum analysis of the electrophysiological data and their comparison to the fluorescent researches date has allowed to assume, that mouse motor nerve terminals are characterized by high rate of endocytosis and fast synaptic vesicle reuse (average recycling time about 50 sec) that can provide effective maintenance of synaptic transmission at long high-frequency activity. Sizes of ready releasable and recycling synaptic vesicle pools are quantitatively determined. It is assumed, that vesicle recycling occurs on a short fast way to inclusion in recycling pool. So, in the stimulation protocol that were used the synaptic vesicles from reserve pool remain unused. Thus in our conditions recycling pool vesicles cycle repeatedly without reserve pool release.     

Streamlined synaptic vesicle cycle in cone photoreceptor terminals

Cone photoreceptors tonically release neurotransmitter in the dark through a continuous cycle of exocytosis and endocytosis. Here, using the synaptic vesicle marker FM1-43, we elucidate specialized features of the vesicle cycle. Unlike retinal bipolar cell terminals, where stimulation triggers bulk membrane retrieval, cone terminals appear to exclusively endocytose small vesicles. These retain their integrity until exocytosis, without pooling their membranes in endosomes. Endocytosed vesicles rapidly disperse through the terminal and are reused with no apparent delay. Unlike other synapses where most vesicles are immobilized and held in reserve, only a small fraction (<15%) becomes immobilized in cones. Photobleaching experiments suggest that vesicles move by diffusion and not by molecular motors on the cytoskeleton and that vesicle movement is not rate limiting for release. The huge reservoir of vesicles that move rapidly throughout cone terminals and the lack of a reserve pool are unique features, providing cones with a steady supply for continuous release.     

Tetanic stimulation recruits vesicles from reserve pool via a cAMP-mediated process in Drosophila synapses

At Drosophila neuromuscular junctions, there are two synaptic vesicle pools, namely the exo/endo cycling pool (ECP) and the reserve pool (RP). We studied the recruitment process from RP using a fluorescent dye, FMI-43. During high-frequency nerve stimulation, vesicles in RP were recruited for release, and endocytosed vesicles were incorporated into both pools, whereas with low-frequency stimulation, vesicles were incorporated into and released from ECP. Release of vesicles from RP was detected electrophysiologically after emptying vesicles in the ECP of transmitter by a H+ pump inhibitor. Recruitment from RP was depressed by inhibitors of steps in the cAMP/PKA cascade and enhanced by their activators. In rutabaga (rut) with low cAMP levels, mobilization of vesicles from RP during tetanic stimulation was depressed, while it was enhanced in dunce (dnc) with high cAMP levels.     

Experimental and modeling investigation of the mechanism of synaptic vesicles recycling

Under the condition of microelectrode recording and fluorescence microscopy with dye FM 1-43 the research of exo-/endocytosis of synaptic vesicles in motor nerve terminals (NT) of frog cutaneous pectoris and white mice diaphragm muscles during high frequency stimulation (20 imp/s) was carried out. A mathematical modeling allowed us to conclude that the obtained experimental data can be explained in the following framework. Three pools of synaptic vesicles are involved in neurotransmitter release in the frog motor NT. Recovery of these pools is provided by endocytosis of two types: fast endocytosis with limited capacity and slow endocytosis. Fast-reconstructing vesicles refill the mobilization pool and slow endocytosis recovers the reserve pool. Our modeling investigation has revealed in frog NT independent recruiting of reserve and mobilization pools to the neurotransmitter secretion, i.e. this pools work concurrently. Experimental data, obtained on mice preparations, are well described with the framework of two-pools model including single type of endocytosis (fast endocytosis).     

Endophilin functions as a membrane-bending molecule and is delivered to endocytic zones by exocytosis

Two models have been proposed for endophilin function in synaptic vesicle (SV) endocytosis. The scaffolding model proposes that endophilin's SH3 domain recruits essential endocytic proteins, whereas the membrane-bending model proposes that the BAR domain induces positively curved membranes. We show that mutations disrupting the scaffolding function do not impair endocytosis, whereas those disrupting membrane bending cause significant defects. By anchoring endophilin to the plasma membrane, we show that endophilin acts prior to scission to promote endocytosis. Despite acting at the plasma membrane, the majority of endophilin is targeted to the SV pool. Photoactivation studies suggest that the soluble pool of endophilin at synapses is provided by unbinding from the adjacent SV pool and that the unbinding rate is regulated by exocytosis. Thus, endophilin participates in an association-dissociation cycle with SVs that parallels the cycle of exo- and endocytosis. This endophilin cycle may provide a mechanism for functionally coupling endocytosis and exocytosis.     

Mobility of synaptic vesicles in different pools in resting and stimulated frog motor nerve terminals

We used fluorescence recovery after photobleaching (FRAP) to measure the mobility of synaptic vesicles in frog motor nerve terminals. Vesicles belonging to the recycling pool or to the reserve pool were selectively labeled with FM1-43. In resting terminals, vesicles in the reserve pool were immobile, while vesicles in the recycling pool were mobile. Nerve stimulation increased the mobility of reserve pool vesicles. Treatment with latrunculin A, which destroyed actin filaments, had no significant effect on mobility, and reducing the temperature likewise had little effect, suggesting that recycling pool vesicles move by simple diffusion. Application of okadaic acid caused vesicle mobility in both pools to increase to the same level. We could model these and others' results quantitatively by taking into account the relative numbers of mobile and immobile vesicles in each pool, and vesicle packing density, which has a large effect on mobility.     

The kinetics of synaptic vesicle pool depletion at CNS synaptic terminals

During sustained action potential (AP) firing at nerve terminals, the rates of endocytosis compared to exocytosis determine how quickly the available synaptic vesicle pool is depleted, in turn influencing presynaptic efficacy. Mechanisms, including rapid kiss-and-run endocytosis as well as local, preferential recycling of docked vesicles, have been proposed as a means to allow endocytosis and recycling to keep up with stimulation. We show here that, for CNS nerve terminals at physiological temperatures, endocytosis is sufficiently fast to avoid vesicle pool depletion during continuous AP firing at 10 Hz. This endocytosis-exocytosis balance persists for turnover of the entire releasable pool of vesicles and allows for efficient escape of FM 4-64, indicating that it is a non-kiss-and-run endocytic event. Thus, under physiological conditions, the sustained speed of vesicle membrane retrieval for the entire releasable pool appears to be sufficiently fast to compensate for exocytosis, avoiding significant vesicle pool depletion during robust synaptic activity.     

The Neurexin/N-Ethylmaleimide-sensitive Factor (NSF) Interaction Regulates Short Term Synaptic Depression

Although Neurexins, which are cell adhesion molecules localized predominantly to the presynaptic terminals, are known to regulate synapse formation and synaptic transmission, their roles in the regulation of synaptic vesicle release during repetitive nerve stimulation are unknown. Here, we show that nrx mutant synapses exhibit rapid short term synaptic depression upon tetanic nerve stimulation. Moreover, we demonstrate that the intracellular region of NRX is essential for synaptic vesicle release upon tetanic nerve stimulation. Using a yeast two-hybrid screen, we find that the intracellular region of NRX interacts with N-ethylmaleimide-sensitive factor (NSF), an enzyme that mediates soluble NSF attachment protein receptor (SNARE) complex disassembly and plays an important role in synaptic vesicle release. We further map the binding sites of each molecule and demonstrate that the NRX/NSF interaction is critical for both the distribution of NSF at the presynaptic terminals and SNARE complex disassembly. Our results reveal a previously unknown role of NRX in the regulation of short term synaptic depression upon tetanic nerve stimulation and provide new mechanistic insights into the role of NRX in synaptic vesicle release.     

Properties of a model of Ca(++)-dependent vesicle pool dynamics and short term synaptic depression

We explore the properties of models of synaptic vesicle dynamics, in which synaptic depression is attributed to depletion of a pool of release-ready vesicles. Two alternative formulations of the model allow for either recruitment of vesicles from an unlimited reserve pool (vesicle state model) or for recovery of a fixed number of release sites to a release-ready state (release-site model). It is assumed that, following transmitter release, the recovery of the release-ready pool of vesicles is regulated by the intracellular free Ca(++) concentration, [Ca(++)](i). Considering the kinetics of [Ca(++)](i) after single presynaptic action potentials, we show that pool recovery can be described by two distinct kinetic components. With such a model, complex kinetic and steady-state properties of synaptic depression as found in several types of synapses can be accurately described. However, the specific assumption that enhanced recovery is proportional to [Ca(++)](i), as measured with Ca(++) indicator dyes, is not confirmed by experiments at the calyx of Held, in which [Ca(++)](i)-homeostasis was altered by adding low concentrations of the exogenous Ca(++) buffer, fura-2, to the presynaptic terminal. We conclude that synaptic depression at the calyx of Held is governed by localized, near membrane [Ca(++)](i) signals not visible to the indicator dye, or else by an altogether different mechanism. We demonstrate that, in models in which a Ca(++)-dependent process is linearly related to [Ca(++)](i), the addition of buffers has only transient but not steady-state consequences.     

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.     

C2B polylysine motif of synaptotagmin facilitates a Ca2+-independent stage of synaptic vesicle priming in vivo

Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca2+ affinity of release, and, at low Ca2+, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca2+ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in syt(null) mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca2+-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca2+-independent docking and/or priming of synaptic vesicles in vivo.     

High- and Low-Mobility Stages in the Synaptic Vesicle Cycle

Synaptic vesicles need to be mobile to reach their release sites during synaptic activity. We investigated vesicle mobility throughout the synaptic vesicle cycle using both conventional and subdiffraction-resolution stimulated emission depletion fluorescence microscopy. Vesicle tracking revealed that recently endocytosed synaptic vesicles are highly mobile for a substantial time period after endocytosis. They later undergo a maturation process and integrate into vesicle clusters where they exhibit little mobility. Despite the differences in mobility, both recently endocytosed and mature vesicles are exchanged between synapses. Electrical stimulation does not seem to affect the mobility of the two types of vesicles. After exocytosis, the vesicle material is mobile in the plasma membrane, although the movement appears to be somewhat limited. Increasing the proportion of fused vesicles (by stimulating exocytosis while simultaneously blocking endocytosis) leads to substantially higher mobility. We conclude that both high- and low-mobility states are characteristic of synaptic vesicle movement.     

Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions

In a forward screen for genes affecting neurotransmission in Drosophila, we identified mutations in dynamin-related protein (drp1). DRP1 is required for proper cellular distribution of mitochondria, and in mutant neurons, mitochondria are largely absent from synapses, thus providing a genetic tool to assess the role of mitochondria at synapses. Although resting Ca2+ is elevated at drp1 NMJs, basal synaptic properties are barely affected. However, during intense stimulation, mutants fail to maintain normal neurotransmission. Surprisingly, FM1-43 labeling indicates normal exo- and endocytosis, but a specific inability to mobilize reserve pool vesicles, which is partially rescued by exogenous ATP. Using a variety of drugs, we provide evidence that reserve pool recruitment depends on mitochondrial ATP production downstream of PKA signaling and that mitochondrial ATP limits myosin-propelled mobilization of reserve pool vesicles. Our data suggest a specific role for mitochondria in regulating synaptic strength.     

Two synaptic vesicle pools,vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction

Drosophila neuromuscular junctions (DNMJs) are malleable and its synaptic strength changes with activities. Mobilization and recruitment of synaptic vesicles (SVs), and replenishment of SV pools in the presynaptic terminal are involved in control of synaptic efficacy. We have studied dynamics of SVs using a fluorescent styryl dye, FM1-43, which is loaded into SVs during endocytosis and released during exocytosis, and identified two SV pools. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low frequency nerve stimulation and releases FM1-43 during exocytosis induced by high K⁺. The ECP locates close to release sites in the periphery of presynaptic boutons. The reserve pool (RP) is loaded and unloaded only during high frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the efficacy of synaptic transmission during low frequency neuronal firing. An increase of cAMP facilitates SV movement from RP to ECP. Post-tetanic potentiation (PTP) correlates well with recruitment of SVs from RP. Neither PTP nor post-tetanic recruitment of SVs from RP occurs in memory mutants that have defects in the cAMP/PKA cascade. Cyotochalasin D slows mobilization of SVs from RP, suggesting involvement of actin filaments in SV movement. During repetitive nerve stimulation the ECP is replenished, while RP replenishment occurs after tetanic stimulation in the absence of external Ca²⁺. Mobilization of internal Ca²⁺ stores underlies RP replenishment. SV dynamics is involved in synaptic plasticity and DNMJs are suitable for further studies.