Fusion of synaptic vesicles and plasma membrane in the presence of synaptosomal soluble proteins

Fusion between synaptic vesicles and plasma membranes isolated from rat brain synaptosomes is regarded as a model of neurosecretion. The main aim of current study is to investigate whether the synaptosomal soluble proteins are essential members of Ca(2+)-triggered fusion examined in this system. Fusion experiments were performed using fluorescent dye octadecylrhodamine B, which was incorporated into synaptic vesicle membranes at self-quenching concentration. The fusion of synaptic vesicles, containing marker octadecylrhodamine B, with plasma membranes was detected by dequenching of the probe fluorescence. Membrane fusion was not found in Ca(2+)-supplemented buffer solution, but was initiated by the addition of the synaptosomal soluble proteins. When soluble proteins were treated with trypsin, they lost completely the fusion activity. These experiments confirmed that soluble proteins of synaptosomes are sensitive to Ca(2+) signal and essential for membrane fusion. The experiments, in which members of fusion process were treated with monoclonal antibodies raised against synaptotagmin and synaptobrevin, have shown that antibodies only partially inhibited fusion of synaptic vesicles and plasma membranes in vitro. These results indicate that other additional component(s), which may or may not be related to synaptobrevin or synaptotagmin, mediate this process. It can be assumed that fusion of synaptic vesicles with plasma membranes in vitro depends upon the complex interaction of a large number of protein factors.     

Ouabain evokes exocytosis dependent on ryanodine and mitochondrial calcium stores that is not followed by compensatory endocytosis at the neuromuscular junction

Ouabain is a cardiotonic glycoside that inhibits the sodium potassium ATPase pump leading to sodium accumulation in nerve terminals. At the frog neuromuscular junction, ouabain induces acetylcholine release and a rapid depletion of synaptic vesicles. In the present work, we used FM1–43 vital labeling to dissect the effect of ouabain on synaptic vesicles recycling. We first examined images of nerve-muscle preparations that were stained with FM1–43 by electrical stimulation of the nerve and destained with ouabain. We observed that ouabain induced exocytosis of synaptic vesicles independently of extracellular calcium, implying a mechanism of exocytosis that can bypass the requirement for extracellular calcium. We therefore tested the hypothesis that ouabain induces exocytosis by mobilizing intracellular calcium and we report that calcium release from endoplasmic reticulum through ryanodine receptors is necessary for ouabain-evoked exocytosis. In addition, the ouabain-evoked exocytosis was dependent on calcium released from mitochondria. We also investigated if exocytosis evoked by ouabain is followed by compensatory endocytosis. We observed that muscles incubated with FM1–43 in the presence of ouabain did not present significant staining. In conclusion, our data demonstrate that exocytosis evoked by ouabain is independent on extracellular calcium but dependent on calcium release from endoplasmic reticulum and mitochondrial stores. In addition, we suggest that ouabain can be used as a pharmacological tool to uncouple synaptic vesicles exocytosis from endocytosis at the neuromuscular junction.     

Nerve growth factor collaborates with myocyte‐derived factors to promote development of presynaptic sites in cultured sympathetic neurons

Nerve growth factor (NGF) acutely modulates synaptic transmission between sympathetic neurons and their cardiac myocyte targets. NGF also has developmental effects in establishing the level of synaptic transmission between sympathetic neurons and myocytes in culture, although little is known about the mechanisms by which NGF influences this synaptic connectivity. Here we report that NGF acts in conjunction with factors produced by cardiac myocytes to promote neuronal contact with the target and the extension of synaptic vesicle‐containing growth cones. In conjunction with previously published results showing that NGF has long‐term effects on synaptic transmission between sympathetic neurons and myocytes, this work suggests that NGF acts to promote sympathetic neurotransmission by increasing the number of sympathetic fibers establishing target contact. Further, we found that developmental changes in cardiac myocytes led to an increase in the density of synaptic vesicle–containing variocosities along sympathetic fibers, a process regulated by NGF. Thus, as myocytes mature they produce factors that promote the formation of sympathetic presynaptic structures. These results argue that multiple target interactions regulate the extent of synapse formation between sympathetic neurons and cardiac cells and suggest that NGF promotes presynaptic development by increasing neuronal contact with myocyte‐derived cell surface or matrix‐associated factors. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 460–476, 2000     

Evidence for early endosome-like fusion of recently endocytosed synaptic vesicles

Early endosomes are well-established acceptor compartments of endocytic vesicles in many cell types. Little evidence of their existence or function has been obtained in synapses, and it is generally believed that synaptic vesicles recycle without passing through an endosomal intermediate. We show here that the early endosomal SNARE proteins are enriched in synaptic vesicles. To investigate their function in the synapse, we isolated synaptic nerve terminals (synaptosomes), stimulated them in presence of different fluorescent markers to label the recycling vesicles and used these vesicles in in vitro fusion assays. The recently endocytosed vesicles underwent homotypic fusion. They also fused with endosomes from PC12 and BHK cells. The fusion process was dependent upon NSF activity. Moreover, fusion was dependent upon the early endosomal SNAREs but not upon the SNAREs involved in exocytosis. Our results thus show that at least a fraction of the vesicles endocytosed during synaptic activity are capable of fusing with early endosomes and lend support to an involvement of endosomal intermediates during recycling of synaptic vesicles.     

The formation of synapses in the central nervous system

Interneuronal synapses are specialized contact zones formed between the transmitting pole of one neuron, usually an axon, and the receptive pole of another nerve cell, usually a dendritic process or the soma. The formation of these synaptic contacts is the result of cellular events related to neurite elongation, the establishment of polarity, axon guidance, and target recognition. A series of morphological rearrangements takes place once synaptic targets establish their initial contact. These changes include the clustering of synaptic vesicles in the presynaptic element and the formation of a specialized area capable of signal transduction at the postsynaptic target. The present review discusses the role of different synaptic proteins in the cellular events leading to the formation of synapses among neurons in the central nervous system.     

Exogenous Glutamate-Induced Modulation of Neurosecretory Process in Nerve Terminals Obtained from the Rat Brain

We studied intracellular processes in nerve terminals of neurons of the rat brain in response to application of exogenous glutamate. Using a рН-sensitive fluorescence probe, acridine orange (AO), and labeled gammaaminobutyric acid ([³Н]GABA), we estimated the effect of application of glutamate on the level of acidification of synaptic vesicles and also on the release of GABA from nerve terminals (synaptosomes) obtained from hippocampal tissue. Our experiments showed that glutamate in a dose-dependent manner stimulated the [³Н]GABA release from nerve terminals, and then we observed re-uptake of this neurotransmitter. A selective blocker of GABA transporters, NO-711, completely blocked the uptake of neurotransmitter but did not influence its release; this observation indicates that the glutamate-induced GABA release was from the vesicular, not cytosolic, pool. We confirmed that glutamate stimulates the process of exocytosis in experiments using AO, where we obtained data indicating that this process is two-phase. The first phase, which reflects probably calcium-induced exocytosis, looked like a “burst” of fluorescent signal typical of the response of synaptosomes to the action of KCl applied in depolarization concentration. Both phases of the response were completely blocked or significantly suppressed in calcium-free medium or in the presence of 25 μM Cd²⁺. The second (slow) phase of the response developed after a certain lag period and was characterized by a gradual increase in the intensity of fluorescent signal. This phase was completely dependent on the presence of sodium in the extracellular medium and completely blocked when sodium was replaced by choline or N-methyl-D-glucamine. We hypothesize that the second phase of the response can reflect either spontaneous unstimulated exocytosis or dissipation of the proton gradient in synaptic vesicles induced by the entry of Na⁺ into the nerve terminal.     

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.     

Okadaic acid disrupts clusters of synaptic vesicles in frog motor nerve terminals

The fluorophore FM1-43 appears to stain membranes of recycled synaptic vesicles. We used FM1-43 to study mechanisms of synaptic vesicle clustering and mobilization in living frog motor nerve terminals. FM1- 43 staining of these terminals produces a linear series of fluorescent spots, each spot marking the cluster of several hundred synaptic vesicles at an active zone. Most agents we tested did not affect staining, but the phosphatase inhibitor okadaic acid (OA) disrupted the fluorescent spots, causing dye to spread throughout the terminal. Consistent with this, electron microscopy showed that vesicle clusters were disrupted by OA treatment. However, dye did not spread passively to a uniform spatial distribution. Instead, time lapse movies showed clear evidence of active dye movements, as if synaptic vesicles were being swept along by an active translocation mechanism. Large dye accumulations sometimes occurred at sites of Schwann cell nuclei. These effects of OA were not significantly affected by pretreatment with colchicine or cytochalasin D. Electrophysiological recordings showed that OA treatment reduced the amount of acetylcholine released in response to nerve stimulation. The results suggest that an increased level of protein phosphorylation induced by OA treatment mobilizes synaptic vesicles and unmasks a powerful vesicle translocation mechanism, which may function normally to distribute synaptic vesicles between active zones.     

Sequential steps of carbohydrate signaling mediate sensory afferent differentiation

Differences in carbohydrate signaling control sequential steps in synaptic growth of sensory afferents in the leech. The relevant glycans are constitutive and developmentally regulated modifications of leechCAM and Tractin (family members of NCAM and L1) that are specific to the surface of sensory afferents. A mannosidic glycosylation mediates the dynamic growth of early afferents as they explore their target region through sprouting sensory arbors rich with synaptic vesicles. Later emerging galactosidic glycosylations serve as markers for subsets of the same sensory afferents that correlate with different sensory modalities. These developmentally regulated galactose markers now oppose the function of the constitutive mannose marker. Sensory afferents gain cell-cell contact with central neurons and self-similar afferents, but lose filopodia and synaptic vesicles. Extant vesicles are confined to sites of en passant synapse formation. The transformation of sensory afferent growth, progressing from mannose- to galactose-specific recognition, is consistent with a change from cell-matrix to cell-cell contact. While the constitutive mannosidic glycosylation promotes dynamic growth, developmentally regulated galactosidic glycosylations of the same cell adhesion molecules promote tissue stability. The persistence of both types of neutral glycans beyond embryonic age allows their function in synaptic plasticity during habituation and learning.     

Extensive segregation of acidic phospholipids in membranes induced by protein kinase C and related proteins

Protein kinase C and two other proteins with molecular masses of 64 and 32 kDa, purified from bovine brain, constitute a type of protein that binds a large number of calcium ions in a phospholipid-dependent manner. This study suggested that these proteins also induced extensive clustering of acidic phospholipids in the membranes. Clustering of acidic phospholipids was detected by the self-quenching of a fluorescence probe that was attached to acidic phospholipids (phosphatidic acid or phosphatidylglycerol). Addition of these proteins to phospholipid vesicles containing 15% fluorescently labeled phosphatidic acid dispersed in neutral phosphatidylcholine resulted in extensive, rapid, and calcium-dependent quenching of the fluorescence signal. Fluorescence-quenching requirements coincided with protein-membrane binding characteristics. As expected, the addition of these proteins to phospholipid vesicles containing fluorescent phospholipids dispersed with large excess of acidic phospholipids produced only small fluorescence changes. In addition, association of these proteins with vesicles composed of 100% fluorescent phospholipids resulted in no fluorescence quenching. Protein binding to vesicles containing 5-50% fluorescent phospholipid showed different levels of fluorescence quenching that closely resemble the behavior expected for extensive segregation of the acidic phospholipids in the outer layer of the vesicles. Thus, the fluorescence quenching appeared to result from self-quenching of the fluorophores that become clustered upon protein-membrane binding. These results were consistent with protein-membrane binding that was maintained by calcium bridges between the proteins and acidic phospholipids in the membrane. Since each protein bound eight or more calcium ions in the presence of phospholipid, they may each induce clustering of a related number of acidic phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Biophysical Characterization of Styryl Dye-Membrane Interactions

Styryl dyes (also referred to as FM dyes) become highly fluorescent upon binding to membranes and are often used to study synaptic vesicle recycling in neurons. To date, however, no direct comparisons of the fluorescent properties, or time-resolved (millisecond) measurements of dye-membrane binding and unbinding reactions, for all members of this family of probes have been reported. Here, we compare the fluorescence intensities of each member of the FM dye family when bound to membranes. This analysis included SGC5, a new lipophilic fluorescent dye with a unique structure. Fluorescence intensities depended on the length of the lipophilic tail of each dye, with a rank order as follows: SGC5 > FM1-84 > FM1-43 > SynaptoGreen C3 > FM2-10/FM4-64/FM5-95. Stopped-flow measurements revealed that dye hydrophobicity determined the affinity and departitioning rates for dye-membrane interactions. All of the dyes dissociated from membranes on the millisecond timescale, which is orders of magnitude faster than the overall destaining rate (timescale of seconds) of these dyes from presynaptic boutons. Departitioning kinetics were faster at higher temperatures, but were unaffected by pH or cholesterol. The data reported here aid interpretation of dye-release kinetics from single synaptic vesicles, and indicate that these probes dissociate from membranes on more rapid timescales than previously appreciated.     

ATP-dependent fusion of synaptic vesicles and synaptosomal plasma membrane in a cell-free system

The final step in exocytosis is the fusion of synaptic vesicle membrane with the synaptosomal plasma membrane, leading to the release of the neurotransmitters. We have reconstituted this fusion event in vitro, using isolated synaptic vesicles and synaptosomal plasma membranes from the bovine brain. The membranes of synaptic vesicles were loaded with the lipid--soluble fluorescent probe octadecylrhodamine B at the concentration that resulted in self-quenching of its fluorescence. The vesicles were then incubated with synaptosomal plasma membranes at 37 degrees C and fusion was measured through the dilution-dependent de-quenching of the fluorescence of the probe. Synaptic vesicles by themselves did not fused with plasma membrane, only addition of ATP induced the fusion. W-7 and trifluoroperasine, the drugs reported to inhibit calmodulin-dependent events, were effective inhibitors of the ATP-induced fusion synaptic vesicles and synaptosomal plasma membranes. Our results indicate that the membrane fusion in the nerve terminals during exocytosis may be under direct control of calmodulin-dependent protein phosphorylation.     

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass¹. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release^2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43®) by co-puffing the dye and a ringer solution containing a high K⁺ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE⁴, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.Open in a separate windowClick here to view.^{(66M, flv)}     

Analysis of living motor nerve ending of a frog by endocytotic fluorescent marker FM 1-43

In our experiments on motor nerve endings of the frog cutaneous pectoris muscle, using fluorescent marker FM 1-43, the intensity and topography of endocytosis were investigated after the initiation of massive exocytosis of synaptic vesicles by increasing the extracellular potassium concentration. Using FM 1-43, fluorescent spots were shown to appear, looking as accumulations of synaptic vesicles in the active zone region. The forms and sizes of luminous spots and the distances between them were analysed. Considerable variations in brightness and total areas of fluorescent spots per a length unit in different regions of the nerve ending were revealed in addition to a proximal-distal gradient of these parameters along the nerve terminal. Peculiarities of topography and intensities of luminescence in the most terminal regions of the nerve ending are described. The obtained data are discussed in terms of the exo- and endocytosis cycle of synaptic vesicles in the active zone region, and from the point of view of the plasticity of the motor nerve ending and active zones. The factors involved in the transmitter release nonuniformity are analysed.     

The ultrastructural interactions of identified pre‐ and postsynaptic cells during synaptic target recognition in Drosophila embryos

During the development of neural networks, what sets synaptogenic interactions apart from nonsynaptogenic interactions is not well understood at the subcellular level. Using a combination of intracellular dye injection and electron microscopy, we show that a specific motoneuron (RP3) and its synaptic partners (muscles 6 and 7), both often bearing microprocesses, develop intimate membrane contact sites characterized by junctional structures, prior to their initiating synaptogenesis in Drosophila embryos. Other motoneuron growth cones that extend alongside the RP3 growth cone to innervate surrounding muscles do not form such contacts with muscles 6 and 7. We also examined how specific target recognition molecules affect the development of these ultrastructural associations between synaptic partner cells. When Fasciclin III (Fas3), a “positive” target recognition molecule for RP3, is ectopically expressed in neighboring muscles, the RP3 growth cone ectopically develops membrane contact sites with Fas3‐misexpressing muscles with which it would not normally associate. In contrast, when Toll, a “negative” target recognition molecule normally expressed by a subset of muscles that surrounds muscles 6 and 7, is misexpressed on muscles 6 and 7, the RP3 growth cone fails to exhibit its normal close contact with these muscles. We propose that the formation of close membrane associations and junctional structures can be regulated under the influence of synaptic target recognition molecules and signifies the beginning of subcellular events during synaptic target recognition. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 448–459, 2000     

Synaptic organization of the cat parietal association cortex (area 5b)

An electron microscopy study was made of synaptic organization in the cat association cortex, area 5b. A total of 1635 axonal terminals were discovered over 6215 µm² (240 electronic imagings of slices of different association cortex layers); i.e., an average of 263±16 terminals per 1000 µm² expanse. It was found that 75.5% of axon terminals contained synaptic vesicles and formed either one- or two-sided contact with postsynaptic structures; 24.5% of axonal terminals contained synaptic vesicles but formed no distinct synaptic contacts with nearby neurons; 84.9% of terminals contained round-shaped or slightly oval synaptic vesicles; 7.8% had both rounded and elongated shapes, and vesicles were very elongated in the remaining 7.3%. Of the axonal terminals having synaptic contacts, axo(dendritic)-spinal terminals accounted for 46.6%, and axodendritic and axosomatic endings amounted to 50.0% and 3.4% respectively (in all 77% of axosomatic terminals contained elongated vesicles and maintained symmetrical contact, while 23% had round-shaped vesicles and formed asymmetrical contact). Calculations show that for each 1 mm³ an average of 258 million axonal terminals are found forming synaptic contacts in the cat association cortex as well as 84 million terminals containing synaptic vesicles but not forming contact.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 174–185, March–April, 1989.     

Calcium and Protein Kinase C Regulate the Actin Cytoskeleton in the Synaptic Terminal of Retinal Bipolar Cells

The organization of filamentous actin (F-actin) in the synaptic pedicle of depolarizing bipolar cells from the goldfish retina was studied using fluorescently labeled phalloidin. The amount of F-actin in the synaptic pedicle relative to the cell body increased from a ratio of 1.6 ± 0.1 in the dark to 2.1 ± 0.1 after exposure to light. Light also caused the retraction of spinules and processes elaborated by the synaptic pedicle in the dark.Isolated bipolar cells were used to characterize the factors affecting the actin cytoskeleton. When the electrical effect of light was mimicked by depolarization in 50 mM K⁺, the actin network in the synaptic pedicle extended up to 2.5 μm from the plasma membrane. Formation of F-actin occurred on the time scale of minutes and required Ca²⁺ influx through L-type Ca²⁺ channels. Phorbol esters that activate protein kinase C (PKC) accelerated growth of F-actin. Agents that inhibit PKC hindered F-actin growth in response to Ca²⁺ influx and accelerated F-actin breakdown on removal of Ca²⁺.To test whether activity-dependent changes in the organization of F-actin might regulate exocytosis or endocytosis, vesicles were labeled with the fluorescent membrane marker FM1-43. Disruption of F-actin with cytochalasin D did not affect the continuous cycle of exocytosis and endocytosis that was stimulated by maintained depolarization, nor the spatial distribution of recycled vesicles within the synaptic terminal. We suggest that the actions of Ca²⁺ and PKC on the organization of F-actin regulate the morphology of the synaptic pedicle under varying light conditions.     

Selective replenishment of two vesicle pools depends on the source of Ca2+ at the Drosophila synapse

After synaptic vesicles (SVs) undergo exocytosis, SV pools are replenished by recycling SVs at nerve terminals. At Drosophila neuromuscular synapses, there are two distinct SV pools (i.e., the exo/endo cycling pool (ECP), which primarily maintains synaptic transmission, and the reserve pool (RP), which participates in synaptic transmission only during tetanic stimulation). Labeling endocytosed vesicular structures with a fluorescent styryl dye, FM1-43, and measuring intracellular Ca2+ concentrations with a Ca2+ indicator, rhod-2, we show here that the ECP is replenished by SVs endocytosed during stimulation, and this process depends on external Ca2+. In contrast, the RP is refilled after cessation of tetanus by a process mediated by Ca2+ released from internal stores.     

Myosin accelerates synaptic vesicle recycling in the motor nerve endings

Neurotransmitter release and exocytosis of synaptic vesicles in the motor nerve endings of the frog cutaneous-pectoris muscle were studied using electrophysiological and optical methods under the conditions of inhibition of the myosin light-chain kinase and non-muscle myosin by the specific inhibitors ML-7 (12 μM) and (–)-blebbistatin (100 μM). At high-frequency stimulation (20 pulses/s), these inhibitors strengthened suppression of transmitter release during the first 20–25 s and slowed down the release of the fluorescent dye FM 1-43. The obtained results indicate that myosin accelerates rapid synaptic vesicle recycling upon high-frequency stimulation.