Kinetics of Synaptic Transmission at Ribbon Synapses of Rods and Cones

The ribbon synapse is a specialized structure that allows photoreceptors to sustain the continuous release of vesicles for hours upon hours and years upon years but also respond rapidly to momentary changes in illumination. Light responses of cones are faster than those of rods and, mirroring this difference, synaptic transmission from cones is also faster than transmission from rods. This review evaluates the various factors that regulate synaptic kinetics and contribute to kinetic differences between rod and cone synapses. Presynaptically, the release of glutamate-laden synaptic vesicles is regulated by properties of the synaptic proteins involved in exocytosis, influx of calcium through calcium channels, calcium release from intracellular stores, diffusion of calcium to the release site, calcium buffering, and extrusion of calcium from the cytoplasm. The rate of vesicle replenishment also limits the ability of the synapse to follow changes in release. Post-synaptic factors include properties of glutamate receptors, dynamics of glutamate diffusion through the cleft, and glutamate uptake by glutamate transporters. Thus, multiple synaptic mechanisms help to shape the responses of second-order horizontal and bipolar cells.     

Synapsin regulation of vesicle organization and functional pools

Synaptic vesicles are organized in clusters, and synapsin maintains vesicle organization and abundance in nerve terminals. At the functional level, vesicles can be subdivided into three pools: the releasable pool, the recycling pool, and the reserve pool, and synapsin mediates transitions between these pools. Synapsin directs vesicles into the reserve pool, and synapsin II isoform has a primary role in this function. In addition, synapsin actively delivers vesicles to active zones. Finally, synapsin I isoform mediates coupling release events to action potentials at the latest stages of exocytosis. Thus, synapsin is involved in multiple stages of the vesicle cycle, including vesicle clustering, maintaining the reserve pool, vesicle delivery to active zones, and synchronizing release events. These processes are regulated via a dynamic synapsin phosphorylation/dephosphorylation cycle which involves multiple phosphorylation sites and several pathways. Different synapsin isoforms have unique and non-redundant roles in the multifaceted synapsin function.     

Vesicle cycle in the presynaptic nerve terminal

Presynaptic nerve terminals contain a great number ofsynaptic vesicles filled with neurotransmitter. The transmission of information in synapses is mediated by release of transmitter from vesicles: exocytosis, after their fusion with presynaptic membrane. At the functioning synapses, the continuous recycling of synaptic vesicles occurs (vesicle cycle), which provides multiple reuse of vesicular membrane material during synaptic activity. Vesicle cycle consists of large number of steps, including vesicle fusion--exocytosis, formation of new vesicles--endocytosis, vesicle sorting, filling of vesicles with transmitter, intraterminal vesicle transport driving the vesicles to different vesicle pools and preparing to next exocytic event. At this paper, I presented the latest literature and our data regarding the steps and mechanisms of vesicle cycle at synapses. Special attention was paid to neuromuscular synapse as the most thoroughly investigated and as my favorite preparation.     

Endocytosis at ribbon synapses

Unlike conventional synaptic terminals that release neurotransmitter episodically in response to action potentials, neurons of the visual, auditory and vestibular systems encode sensory information in graded signals that are transmitted at their synapses by modulating the rate of continuous release. The synaptic ribbon, a specialized structure found at the active zones of these neurons, is necessary to sustain the high rates of exocytosis required for continuous release. To maintain the fidelity of synaptic transmission, exocytosis must be balanced by high-capacity endocytosis, to retrieve excess membrane inserted during vesicle fusion. Capacitance measurements following vesicle release in ribbon-type neurons indicate two kinetically distinct phases of compensatory endocytosis, whose relative contributions vary with stimulus intensity. The two phases can be independently regulated and may reflect different underlying mechanisms operating on separate pools of recycling vesicles. Electron microscopy shows diversity among ribbon-type synapses in the relative importance of clathrin-mediated endocytosis versus bulk membrane retrieval as mechanisms of compensatory endocytosis. Ribbon synapses, like conventional synapses, make use of multiple endocytosis pathways to replenish synaptic vesicle pools, depending on the physiological needs of the particular cell type.     

Synapsins as regulators of neurotransmitter release

One of the crucial issues in understanding neuronal transmission is to define the role(s) of the numerous proteins that are localized within presynaptic terminals and are thought to participate in the regulation of the synaptic vesicle life cycle. Synapsins are a multigene family of neuron-specific phosphoproteins and are the most abundant proteins on synaptic vesicles. Synapsins are able to interact in vitro with lipid and protein components of synaptic vesicles and with various cytoskeletal proteins, including actin. These and other studies have led to a model in which synapsins, by tethering synaptic vesicles to each other and to an actin-based cytoskeletal meshwork, maintain a reserve pool of vesicles in the vicinity of the active zone. Perturbation of synapsin function in a variety of preparations led to a selective disruption of this reserve pool and to an increase in synaptic depression, suggesting that the synapsin-dependent cluster of vesicles is required to sustain release of neurotransmitter in response to high levels of neuronal activity. In a recent study performed at the squid giant synapse, perturbation of synapsin function resulted in a selective disruption of the reserve pool of vesicles and in addition, led to an inhibition and slowing of the kinetics of neurotransmitter release, indicating a second role for synapsins downstream from vesicle docking. These data suggest that synapsins are involved in two distinct reactions which are crucial for exocytosis in presynaptic nerve terminals. This review describes our current understanding of the molecular mechanisms by which synapsins modulate synaptic transmission, while the increasingly well-documented role of the synapsins in synapse formation and stabilization lies beyond the scope of this review.     

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.     

A Highlights from MBoC Selection: Synaptic neuropeptide release by dynamin-dependent partial release from circulating vesicles

Neurons release neuropeptides, enzymes, and neurotrophins by exocytosis of dense-core vesicles (DCVs). Peptide release from individual DCVs has been imaged in vitro with endocrine cells and at the neuron soma, growth cones, neurites, axons, and dendrites but not at nerve terminals, where peptidergic neurotransmission occurs. Single presynaptic DCVs have, however, been tracked in native terminals with simultaneous photobleaching and imaging (SPAIM) to show that DCVs undergo anterograde and retrograde capture as they circulate through en passant boutons. Here dynamin (encoded by the shibire gene) is shown to enhance activity-evoked peptide release at the Drosophila neuromuscular junction. SPAIM demonstrates that activity depletes only a portion of a single presynaptic DCV''s content. Activity initiates exocytosis within seconds, but subsequent release occurs slowly. Synaptic neuropeptide release is further sustained by DCVs undergoing multiple rounds of exocytosis. Synaptic neuropeptide release is surprisingly similar regardless of anterograde or retrograde DCV transport into boutons, bouton location, and time of arrival in the terminal. Thus vesicle circulation and bidirectional capture supply synapses with functionally competent DCVs. These results show that activity-evoked synaptic neuropeptide release is independent of a DCV''s past traffic and occurs by slow, dynamin-dependent partial emptying of DCVs, suggestive of kiss-and-run exocytosis.     

Encoding light intensity by the cone photoreceptor synapse

How cone synapses encode light intensity determines the precision of information transmission at the first synapse on the visual pathway. Although it is known that cone photoreceptors hyperpolarize to light over 4-5 log units of intensity, the relationship between light intensity and transmitter release at the cone synapse has not been determined. Here, we use two-photon microscopy to visualize release of the synaptic vesicle dye FM1-43 from cone terminals in the intact lizard retina, in response to different stimulus light intensities. We then employ electron microscopy to translate these measurements into vesicle release rates. We find that from darkness to bright light, release decreases from 49 to approximately 2 vesicles per 200 ms; therefore, cones compress their 10,000-fold operating range for phototransduction into a 25-fold range for synaptic vesicle release. Tonic release encodes ten distinguishable intensity levels, skewed to most finely represent bright light, assuming release obeys Poisson statistics.     

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.     

A membrane marker leaves synaptic vesicles in milliseconds after exocytosis in retinal bipolar cells

Perhaps synaptic vesicles can recycle so rapidly because they avoid complete exocytosis, and release transmitter through a fusion pore that opens transiently. This view emerges from imaging whole terminals where the fluorescent lipid FM1-43 seems unable to leave vesicles during transmitter release. Here we imaged single, FM1-43-stained synaptic vesicles by evanescent field fluorescence microscopy, and tracked the escape of dye from single vesicles by watching the increase in fluorescence after exocytosis. Dye left rapidly and completely during most or all exocytic events. We conclude that vesicles at this terminal allow lipid exchange soon after exocytosis, and lose their dye even if they connected with the plasma membrane only briefly. At the level of single vesicles, therefore, observations with FM1-43 provide no evidence that exocytosis of synaptic vesicles is incomplete.     

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.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.     

Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals

We have identified and characterized two vesicle recycling pathways in frog motor nerve terminals. We exploited the differential staining properties of FM dyes of varying hydrophobicity to label selectively two different vesicle pools, using optical imaging and electron microscopy of photoconverted dyes. During a 1 min tetanus, a rapidly recycling route places vesicles selectively into a small readily releasable pool comprising about 20% of vesicles. After the tetanus, a much slower pathway (from which FM2-10 but not FM1-43 can be rinsed) delivers vesicles via infoldings and cisternae selectively to a reserve pool with a halftime of about 8 min. Mixing between the two pools is slow. During stimulation at 30 Hz, 10-15 s is required to mobilize and release dye from the reserve pool.     

Synapsin is a novel Rab3 effector protein on small synaptic vesicles. I. Identification and characterization of the synapsin I-Rab3 interactions in vitro and in intact nerve terminals

Synapsins, a family of neuron-specific phosphoproteins, have been demonstrated to regulate the availability of synaptic vesicles for exocytosis by binding to both synaptic vesicles and the actin cytoskeleton in a phosphorylation-dependent manner. Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a reserve pool of synaptic vesicles, recent results suggest that the synapsins may also be involved in some later step of exocytosis. In order to investigate additional interactions of the synapsins with nerve terminal proteins, we have employed phage display library technology to select peptide sequences binding with high affinity to synapsin I. Antibodies raised against the peptide YQYIETSMQ (syn21) specifically recognized Rab3A, a synaptic vesicle-specific small G protein implicated in multiple steps of exocytosis. The interaction between synapsin I and Rab3A was confirmed by photoaffinity labeling experiments on purified synaptic vesicles and by the formation of a chemically cross-linked complex between synapsin I and Rab3A in intact nerve terminals. Synapsin I could be effectively co-precipitated from synaptosomal extracts by immobilized recombinant Rab3A in a GTP-dependent fashion. In vitro binding assays using purified proteins confirmed the binding preference of synapsin I for Rab3A-GTP and revealed that the COOH-terminal regions of synapsin I and the Rab3A effector domain are required for the interaction with Rab3A to occur. The data indicate that synapsin I is a novel Rab3 interactor on synaptic vesicles and suggest that the synapsin-Rab3 interaction may participate in the regulation of synaptic vesicle trafficking within the nerve terminals.     

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.     

Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system

Following the fusion of synaptic vesicles with the presynaptic plasma membrane of nerve terminals by the process of exocytosis, synaptic-vesicle components are recycled to replenish the vesicle pool. Here we use a pH-sensitive green fluorescent protein to measure the residence time of VAMP, a vesicle-associated SNARE protein important for membrane fusion, on the surfaces of synaptic terminals of hippocampal neurons following exocytosis. The time course of VAMP retrieval depends linearly on the amount of VAMP that is added to the plasma membrane, with retrieval occurring between about 4 seconds and 90 seconds after exocytosis, and newly internalized vesicles are rapidly acidified. These data are well described by a model in which endocytosis appears to be saturable, but proceeds with an initial maximum velocity of about one vesicle per second. We also find that, following exocytosis, a portion of the newly inserted VAMP appears on the surface of the axon.     

Cholesterol as a key player in the balance of evoked and spontaneous glutamate release in rat brain cortical synaptosomes

Membrane rafts are domains enriched in sphingolipids, glycolipids and cholesterol that are able to compartmentalize cellular processes. Noteworthy, many proteins have been assigned to membrane rafts including those related to the control of the synaptic vesicle release machinery, which is a important step for neurotransmission between synapses. In this work, we have investigated the role of cholesterol in key steps of glutamate release in isolated nerve terminals (synaptosomes) from rat brain cortices. Incubation of synaptosomes with methyl-β-cyclodextrin (MβCD) induced glutamate release in a dose-dependent fashion. HγCD, a cyclodextrin with low affinity for cholesterol, had no significant effect on spontaneous glutamate release. When we evaluated the effects of MβCD on glutamate release induced by depolarizing stimuli, we observed that MβCD treatment inhibited the KCl-evoked glutamate release. The glutamate release induced by MβCD was not altered by treatment with EGTA nor with EGTA-AM. The KCl-evoked glutamate release was no further inhibited when synaptosomes were incubated with MβCD in the absence of calcium. We therefore investigated whether the cholesterol removal by MβCD changes intrasynaptosomal sodium and calcium levels. Our results suggested that the cholesterol removal effect on spontaneous and evoked glutamate release might be upstream to sodium and calcium entry through voltage-activated channels. We therefore tested if MβCD would have a direct effect on synaptic vesicle exocytosis and we showed that cholesterol removal by MβCD induced spontaneous exocytosis and inhibited synaptic vesicle exocytosis evoked by depolarizing stimuli. Lastly, we investigated the effect of protein kinase inhibitors on the spontaneous exocytosis evoked by MβCD and we observed a statistically significant reduction of synaptic vesicles exocytosis. In conclusion, our work shows that cholesterol removal facilitates protein kinase activation that favors spontaneous synaptic vesicles and consequently glutamate release in isolated nerve terminals.     

Properties of synaptic vesicle pools in mature central nerve terminals

Readily releasable and reserve pools of synaptic vesicles play different roles in neurotransmission, and it is important to understand their recycling and interchange in mature central synapses. Using adult rat cerebrocortical synaptosomes, we have shown that 100 mosm hypertonic sucrose caused complete exocytosis of only the readily releasable pool (RRP) of synaptic vesicles containing glutamate or gamma-aminobutyric acid. Repetitive hypertonic stimulations revealed that this pool recycled (and reloaded the neurotransmitter from the cytosol) fully in <30 s and did so independently of the reserve pool. Multiple rounds of exocytosis could occur in the constant absence of extracellular Ca(2+). However, although each vesicle cycle includes a Ca(2+)-independent exocytotic step, some other stage(s) critically require an elevation of cytosolic [Ca(2+)], and this is supplied by intracellular stores. Repetitive recycling also requires energy, but not the activity of phosphatidylinositol 4-kinase, which maintains the normal level of phosphoinositides. By varying the length of hypertonic stimulations, we found that approximately 70% of the RRP vesicles fused completely with the plasmalemma during exocytosis and could then enter silent pools, probably outside active zones. The rest of the RRP vesicles underwent very fast local recycling (possibly by kiss-and-run) and did not leave active zones. Forcing the fully fused RRP vesicles into the silent pool enabled us to measure the transfer of reserve vesicles to the RRP and to show that this process requires intact phosphatidylinositol 4-kinase and actin microfilaments. Our findings also demonstrate that respective vesicle pools have similar characteristics and requirements in excitatory and inhibitory nerve terminals.     

The Molecular Architecture of Ribbon Presynaptic Terminals

The primary receptor neurons of the auditory, vestibular, and visual systems encode a broad range of sensory information by modulating the tonic release of the neurotransmitter glutamate in response to graded changes in membrane potential. The output synapses of these neurons are marked by structures called synaptic ribbons, which tether a pool of releasable synaptic vesicles at the active zone where glutamate release occurs in response to calcium influx through L-type channels. Ribbons are composed primarily of the protein, RIBEYE, which is unique to ribbon synapses, but cytomatrix proteins that regulate the vesicle cycle in conventional terminals, such as Piccolo and Bassoon, also are found at ribbons. Conventional and ribbon terminals differ, however, in the size, molecular composition, and mobilization of their synaptic vesicle pools. Calcium-binding proteins and plasma membrane calcium pumps, together with endomembrane pumps and channels, play important roles in calcium handling at ribbon synapses. Taken together, emerging evidence suggests that several molecular and cellular specializations work in concert to support the sustained exocytosis of glutamate that is a hallmark of ribbon synapses. Consistent with its functional importance, abnormalities in a variety of functional aspects of the ribbon presynaptic terminal underlie several forms of auditory neuropathy and retinopathy.     

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.