Mechanisms of synaptic vesicle recycling illuminated by fluorescent dyes

The recycling of synaptic vesicles in nerve terminals involves multiple steps, underlies all aspects of synaptic transmission, and is a key to understanding the basis of synaptic plasticity. The development of styryl dyes as fluorescent molecules that label recycling synaptic vesicles has revolutionized the way in which synaptic vesicle recycling can be investigated, by allowing an examination of processes in neurons that have long been inaccessible. In this review, we evaluate the major aspects of synaptic vesicle recycling that have been revealed and advanced by studies with styryl dyes, focussing upon synaptic vesicle fusion, retrieval, and trafficking. The greatest impact of styryl dyes has been to allow the routine visualization of endocytosis in central nerve terminals for the first time. This has revealed the kinetics of endocytosis, its underlying sequential steps, and its regulation by Ca2+. In studies of exocytosis, styryl dyes have helped distinguish between different modes of vesicle fusion, provided direct support for the quantal nature of exocytosis and endocytosis, and revealed how the probability of exocytosis varies enormously from one nerve terminal to another. Synaptic vesicle labelling with styryl dyes has helped our understanding of vesicle trafficking by allowing better understanding of different synaptic vesicle pools within the nerve terminal, vesicle intermixing, and vesicle clustering at release sites. Finally, the dyes are now being used in innovative ways to reveal further insights into synaptic plasticity.     

Recycling of Synaptic Vesicles at the Frog Neuromuscular Junction in the Presence of Strontium

Abstract: In these experiments, we followed the exocytosis and endocytosis of synaptic vesicles with the vital dye FM1-43 and asked whether calcium is important for membrane retrieval at the frog neuromuscular junction. We replaced calcium with equimolar amounts of strontium and monitored the staining of recycling vesicles by inducing exocytosis with electrical stimulation. Trains of 2,400 (2 or 20 Hz) or 4,200 (20 Hz) pulses failed to induce FM1-43 internalization in the presence of strontium, but they did in the presence of calcium. This effect of strontium was not due to a decrease in exocytosis, because FM1-43 release was similar in the presence of calcium or strontium. The impairment in endocytosis, observed as inhibition of FM1-43 internalization, could be overcome by longer periods of stimulation (6,000 pulses at 2 or 20 Hz) in the presence of strontium (1.8 m M ) or by increasing the extracellular concentration of strontium to 10 m M (2,400 action potentials at 20 Hz). It is suggested that endocytosis is dependent on calcium influx and that strontium is much less effective in replacing calcium for endocytosis than it is for exocytosis.     

神经节内板状末梢是豚鼠食道迷走传入神经末梢的机械敏感性受体

电生理学研究发现迷走传入神经在胃肠道的特有结构——神经节内板状末梢(intraganglionic laminar endings,IGLEs)具有感受机械刺激的功能,推断其为迷走神经机械敏感性受体。但是电生理学方法不能将IGLEs的特异结构与其感受机械刺激的功能同时显示出来,而且IGLEs作为机械敏感性受体,其传导机械刺激的机制尚不清楚。本研究应用活性依赖性荧光染料 FM1-43结合牵拉刺激豚鼠食道显示激活的IGLEs结构,以期观察IGLEs是否对机械刺激敏感。同时用多种药物阻断或促进豚鼠食道IGLEs的激活以探讨IGLEs传导机械刺激的机制。应用神经顺行标记技术以验证FM1-43显示的特异结构是否为IGLEs。结果表明,牵拉刺激结合FM1-43染色显示的结构与神经顺行标记法一致,牵拉刺激组激活的IGLEs数目明显多于未牵拉组 [(90．4±9．5)％vs(10．7±2．1)％,P<0．05]。IGLEs对牵拉刺激的敏感性,表明IGLEs是迷走传入神经在胃肠道内感受机械刺激的受体。TTX,阿托品和钙离子对牵拉刺激激活IGLEs无明显影响,表明IGLEs对机械刺激的传导不需要神经递质以及动作电位的传导,而是直接通过机械门控离子通道实现的。多种TRP通道阻断剂包括SKF,gadolinium对IGLEs的激活无影响,而上皮钠离子通道阻断剂benzamil可以明显阻断IGLEs的激活,因此推断,IGLEs结构中传导机械刺激的离子通道可能属于上皮钠离子通道家族而非电压门控钠离子通道或TRP通道。     

Preserved synaptic vesicle recycling in hippocampal neurons in a mouse Alzheimer's disease model

A recently described triple-transgenic mouse model (3xTg, PS1(M146V), APP(Swe), and tau(P301L)) develops a neuropathology similar to the brains of Alzheimer's disease patients including progressive deposits of plaques and tangles [Neuron 39 (2003) 409]. These mice also show age-related deficits in hippocampal synaptic plasticity that occurs before the development of plaques and tangles. Here we report unchanged synaptic vesicle recycling, as measured by FM1-43 release, in the hippocampal neurons of the 3xTg mice. Expression levels of presynaptic protein synaptophysin and of proteins involved in synaptic vesicle recycling including AP180, dynamin I, and synaptotagmin I also remain unaffected. These data suggest that the synaptic deficits observed in the 3xTg neurons may not arise from the preserved synaptic vesicle recycling.     

Evidence for sustained ATP release from liver cells that is not mediated by vesicular exocytosis

Extracellular ATP regulates many important cellular functions in the liver by stimulating purinergic receptors. Recent studies have shown that rapid exocytosis of ATP-enriched vesicles contributes to ATP release from liver cells. However, this rapid ATP release is transient, and ceases in ~30 s after the exposure to hypotonic solution. The purpose of these studies was to assess the role of vesicular exocytosis in sustained ATP release. An exposure to hypotonic solution evoked sustained ATP release that persisted for more than 15 min after the exposure. Using FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide) fluorescence to measure exocytosis, we found that hypotonic solution stimulated a transient increase in FM1-43 fluorescence that lasted ~2 min. Notably, the rate of FM1-43 fluorescence and the magnitude of ATP release were not correlated, indicating that vesicular exocytosis may not mediate sustained ATP release from liver cells. Interestingly, mefloquine potently inhibited sustained ATP release, but did not inhibit an increase in FM1-43 fluorescence evoked by hypotonic solution. Consistent with these findings, when exocytosis of ATP-enriched vesicles was specifically stimulated by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), mefloquine failed to inhibit ATP release evoked by NPPB. Thus, mefloquine can pharmacologically dissociate sustained ATP release and vesicular exocytosis. These results suggest that a distinct mefloquine-sensitive membrane ATP transport may contribute to sustained ATP release from liver cells. This novel mechanism of membrane ATP transport may play an important role in the regulation of purinergic signaling in liver cells.     

Dynamin1 在应激致海马损伤中作用的初步探讨

Dynamin1是Dynamin 家族中的一员,它在突触囊泡的内吞和循环过程中起着重要作用,但Dynamin 1 在应激致海马损伤过程中是否发挥作用还未见报道。为了初步探讨Dynamin 1 在应激致海马损伤中的作用,分别 建立了海马应激损伤的动物模型和细胞模型来进行研究,研究结果表明,随着应激强度的增大,大鼠海马长时程增强(long-term potentiation,LTP) 效应逐渐减弱,同时通过细胞模型FM1-43 荧光染色检测到海马神经元的内吞作用也逐渐减弱,而这两个模型中Dynamin1 的表达也分别相应地下降。这些结果提示：应激过程导致了
Dynamin1 表达水平的下降,进而造成突触囊泡的内吞过程受阻,致使海马LTP效应减弱,并最终导致海马学习记忆功能衰退。     

Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins

The reactive species peroxynitrite, formed via the near diffusion-limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc-18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1-43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO(3), indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate-resistant SNARE complex in a dose-dependent manner (100-1000 microm) and induced the formation of 3-nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein-protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up-regulate neuronal signaling.     

The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane

The mechanism whereby cAMP stimulates Cl(-) flux through CFTR ion channels in secretory epithelia remains controversial. It is generally accepted that phosphorylation by cAMP-dependent protein kinase increases the open probability of the CFTR channel. A more controversial hypothesis is that cAMP triggers the translocation of CFTR from an intracellular pool to the cell surface. We have monitored membrane turnover in Calu-3 cells, a cell line derived from human airway submucosal glands that expresses high levels of CFTR using membrane capacitance and FM1-43 fluorescence measurements. Using a conventional capacitance measurement technique, we observe an apparent increase in membrane capacitance in most cells that exhibit an increase in Cl(-) current. However, after we carefully correct our recordings for changes in membrane conductance, the apparent changes in capacitance are eliminated. Measurements using the fluorescent membrane marker FM1-43 also indicate that no changes in membrane turnover accompany the activation of CFTR. Robust membrane insertion can be triggered with photorelease of caged Ca(2)+ in Calu-3 cells. However, no increase in Cl(-) current accompanies Ca(2)+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca(2)+ lead to transport of CFTR to the plasma membrane in Calu-3 cells. In addition, we conclude that membrane capacitance measurements must be interpreted with caution when large changes in membrane conductance occur.     

Localization of the Extracellular Matrix Protein SC1 Coincides with Synaptogenesis During Rat Postnatal Development

SC1 is an extracellular matrix protein that belongs to the SPARC family of matricellular molecules. This anti-adhesive protein localizes to synapses in the adult rat brain and has been postulated to modulate synapse shape. In this study, increased levels of SC1 were detected from postnatal days 10–20, with a peak at postnatal day 15, a period of intense synaptogenesis. During this time, increased colocalization of SC1 with the synaptic marker synaptophysin was observed in synapse-rich regions of the cerebellum and the cerebral cortex. These findings indicate that the pattern of SC1 localization coincided with synaptogenesis during rat postnatal development.     

Evidence for AMPK-dependent regulation of exocytosis of lipoproteins in a model liver cell line

5′-AMP-activated kinase (AMPK) plays a key role in the regulation of cellular lipid metabolism. The contribution of vesicular exocytosis to this regulation is not known. Accordingly, we studied the effects of AMPK on exocytosis and intracellular lipid content in a model liver cell line. Activation of AMPK by metformin or 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) increased the rates of constitutive exocytosis by about 2-fold. Stimulation of exocytosis by AMPK occurred within minutes, and persisted after overnight exposure to metformin or AICAR. Activation of AMPK also increased the amount of triacylglycerol (TG) and apolipoprotein B (apoB) secreted from lipid-loaded cells. These effects were accompanied by a decrease in the intracellular lipid content indicating that exocytosis of lipoproteins was involved in these lipid-lowering effects. While AMPK increased the rates of fatty acid oxidation (FAO), the lipid-lowering effects were quantitatively significant even after inhibition of FAO with R-etomoxir. These results suggest that hepatic AMPK stimulates constitutive exocytosis of lipoproteins, which may function in parallel with FAO to regulate intracellular lipid content.     

Okadaic acid disrupts synaptic vesicle trafficking in a ribbon-type synapse

Protein phosphorylation plays an essential role in regulating synaptic transmission and plasticity. However, regulation of vesicle trafficking towards and away from the plasma membrane is poorly understood. Furthermore, the extent to which phosphorylation modulates ribbon-type synapses is unknown. Using the phosphatase inhibitor okadaic acid (OA), we investigated the influence of persistent phosphorylation on vesicle cycling in goldfish bipolar cells. We followed uptake of FM1-43 during vesicle recycling in control and OA-treated cells. FM1-43 fluorescence spread to the center of control synaptic terminals after depolarization elicited Ca2+ influx. However, OA (1-50 nm) impaired this spatial spread of FM1-43 in a dose-dependent manner. Capacitance measurements revealed that OA (50 nm) did not modify either the amount or kinetics of exocytosis and endocytosis evoked by depolarizing pulses. The extremely low concentrations of OA (1-5 nm) sufficient to observe the inhibition of vesicle mobility implicate phosphatase 2A (PP2A) as a major regulator of vesicle trafficking after endocytosis. These results contrast with those at the neuromuscular junction where OA enhances lateral movement of vesicles between distinct vesicle clusters. Thus, our results suggest that phosphatases regulate vesicle translocation at ribbon synapses in a different manner than conventional active zones.     

Differential regulation of granule-to-granule and granule-to-plasma membrane fusion during secretion from rat pituitary lactotrophs

We used fluorescence imaging of individual exocytic events together with electron microscopy to study the regulation of dense core granule-to-plasma membrane fusion and granule-to-granule fusion events that occur during secretion from rat pituitary lactotrophs. Stimulating secretion with elevated extracellular potassium, with the calcium ionophore ionomycin, or with thyrotropin releasing hormone or vasoactive intestinal polypeptide resulted in abundant exocytic structures. Approximately 67% of these structures consisted of multiple granules fused together sharing a single exocytic opening with the plasma membrane, i.e., compound exocytosis. For all of these stimulation conditions there appeared to be a finite number of plasma membrane fusion sites, approximately 11 sites around each cellular equator. However, a granule could fuse directly with another granule that had already fused with the plasma membrane even before all plasma membrane sites were occupied. Granule-to-plasma membrane and granule-to-granule fusion events were subject to different regulations. Forskolin, which can elevate cAMP, increased the number of granule-to-granule fusion events without altering the number of granule-to-plasma membrane fusion events. In contrast, the phorbol ester PMA, which activates protein kinase C increased both granule-to-granule and granule-to-plasma membrane fusion events. These results provide a cellular mechanism that can account for the previously demonstrated potentiation of secretion from lactotrophs by cAMP- and PKC-dependent pathways.     

Four-dimensional imaging of transvacuolar strand dynamics in tobacco BY-2 cells

The vacuole is a characteristic organelle of plant cells and fulfills several important functions related to metabolism and growth of the cell. To shed light on the details of vacuolar structural changes in plant cells, we explored the three-dimensional organization and dynamics of living Nicotiana tabacum L. cv. Bright Yellow 2 cell vacuoles by real-time confocal time-lapse imaging. For imaging, the cells were pulse-labeled with the amphipathic styryl dye FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide), which is delivered to the plant vacuole by endocytic uptake and then incubated overnight. Imaging of the membrane-labeled vacuole revealed a complex vacuole morphology underlaid by constant remodeling. The vacuole is traversed by multiple transvacuolar strands which move along each other and fuse in multiple manners. New strands were created by fission of large membrane sheets. Endocytic vesicle trafficking was followed within the dynamic transvacuolar strands. The movement occurred in a stop-and-go fashion with an average vesicle velocity of 0.46 microm/s and a peak velocity of 0.82 microm/s. Transvacuolar-strand reduction and creation is a characteristic event observed during mitosis. Here we propose a mechanistic model for the alteration of the number of transvacuolar strands, on the basis of their fusion and fission.     

Calcium Dependence of Synaptic Vesicle Recycling Before and After Synaptogenesis

Abstract: Using an immunocytochemical assay to monitor synaptic vesicle exocytosis/endocytosis independently of neurotransmitter release, we have investigated some aspects of vesicle recycling in hippocampal neurons at different developmental stages. A calcium- and depolarization-dependent exocytotic/endocytotic recycling of synaptic vesicles was found to take place in neurons already before the formation of synaptic contacts. The analysis of synaptic vesicle recycling at different calcium concentrations revealed the presence of two release components: the first one activated by low calcium concentrations and sustaining vesicle recycling before synaptogenesis, and a second one activated by high calcium concentrations, which is specifically turned on after the establishment of synaptic contacts. These data suggest that formation of synapses correlates with the activation of a putative low-affinity calcium sensor, which allows synaptic vesicle exocytosis to be triggered and turned off over extremely short time scales, in response to large increases in the level of intracellular calcium.