Single vesicle tracking for studying synaptic vesicle dynamics in small central synapses

Sustained neurotransmission is driven by a continuous supply of synaptic vesicles to the release sites and modulated by synaptic vesicle dynamics. However, synaptic vesicle dynamics in synapses remain elusive because of technical limitations. Recent advances in fluorescence imaging techniques have enabled the tracking of single synaptic vesicles in small central synapses in living neurons. Single vesicle tracking has uncovered a wealth of new information about synaptic vesicle dynamics both within and outside presynaptic terminals, showing that single vesicle tracking is an effective tool for studying synaptic vesicle dynamics. Particularly, single vesicle tracking with high spatiotemporal resolution has revealed the dependence of synaptic vesicle dynamics on the location, stages of recycling, and neuronal activity. This review summarizes the recent findings from single synaptic vesicle tracking in small central synapses and their implications in synaptic transmission and pathogenic mechanisms of neurodegenerative diseases.     

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.     

Intermolecular disulfide bonds at central nervous system synaptic junctions

Most proteins in isolated synaptic junctions and nearly all those in postsynaptic densities (the fibrous protein matrix underlying the postsynaptic membrane at the synapse) are extensively cross-linked by disulfide bonds into polymers with a molecular weight of 350,000 or greater. Since the postsynaptic density appears to consist primarily of a matrix of cytoplasmic proteins, such as tubulin and neurofilament protein, our results indicate that at the membrane such proteins may use disulfide bonds to differentiate into the postsynaptic density and tie into the postsynaptic membrane.     

Differential expression of endophilin 1 and 2 dimers at central nervous system synapses

Endophilin 1 is proposed to participate in synaptic vesicle biogenesis through SH3 domain-mediated interactions with the polyphosphoinositide phosphatase synaptojanin and the GTPase dynamin. Endophilin family members have also been identified as binding partners for a number of diverse cellular proteins. We define here the endophilin 1-binding site within synaptojanin 1 and show that this sequence independently and selectively purifies from brain extracts endophilin 1 and a closely related protein, endophilin 2. Endophilin 2, like endophilin 1, is highly expressed in brain, concentrated in nerve terminals, and found in complexes with synaptojanin and dynamin. Although a fraction of endophilins 1 and 2 coexist in the same complex, the distribution of these endophilin isoforms among central synapses only partially overlaps. Endophilins 1 and 2 are found predominantly as stable dimers through a predicted coiled-coil domain in their conserved NH2-terminal moiety. Dimerization may allow endophilins to link a number of different cellular targets to the endocytic machinery.     

Lipid regulation of the synaptic vesicle cycle

Membrane vesicle cycling is orchestrated through the combined actions of proteins and lipids. At neuronal synapses, this orchestration must meet the stringent demands of speed, fidelity and sustainability of the synaptic vesicle cycle that mediates neurotransmission. Historically, the lion's share of the attention has been focused on the proteins that are involved in this cycle; but, in recent years, it has become clear that the previously unheralded plasma membrane and vesicle lipids are also key regulators of this cycle. This article reviews recent insights into the roles of lipid-modifying enzymes and lipids in the acute modulation of neurotransmission.     

Carbohydrate composition of central nervous system synapses: Analysis of isolated synaptic junctional complexes and postsynaptic densities

The composition of specialized structures present at synapses within the central nervous system was elucidated by biochemical analysis of fractions enriched in synaptic junctional complexes and postsynaptic densities. The results indicate that the synaptic junctional complex is primarily protein together with some glycoproteins. The synaptic junctional complex proteins are similar in amino acid composition to synaptic membrane proteins; they are not especially rich in basic residues, as previously suggested. The major carbohydrates present in the synaptic junctional complex and postsynaptic density glycoproteins are mannose, galactose, and glucosamine, with lesser amounts of fucose, $\text{N-}\text{acetylneuraminic}$ acid, and galactosamine. Comparison with the synaptic membrane fraction indicates that galactose is more concentrated in the synaptic junctional complex and mannose in the postsynaptic density. Glucose is dramatically enriched in both these fractions. Sucrose binding during isolation may partially account for the glucose enrichment.     

Cholinergic transmission via central synapses in the locust nervous system

In this study we examine the nature of chemical synaptic transmission between identified filiform hair receptors on the prothoracic segment of a locust and the identified postsynaptic projection interneuron (A4I1). The effects of pressure ejected acetylcholine, and various ligands of acetylcholine receptors on the activity of the postsynaptic neuron A4I1, or on wind-elicited responses in A4I1 are reported. It is suggested that the transmitter of the afferent fibers is acetylcholine, and that fast transmission is mediated by nicotinic acetylcholine-receptors. Both nicotine and carbachol act as agonists, whereas d-tubocurarine and alpha-bungarotoxin act as antagonists. The presence of muscarinic acetylcholine receptors was also evident from the modulatory effects of muscarine, oxotremorine and pilocarpine, which were blocked by bath application of atropine. GABA, and its agonists muscimol and cis-4-amino-crotonic-acid lead to inhibition of A4I1 responses. This inhibition was prevented by the additional application of picrotoxin. This suggests involvement of a ligand-gated GABA receptor which, most likely, increases chloride conductance. Metabotropic GABA-receptors do not seem to be involved, since baclofene, diazepam and bicuculline ejections had no effects. Glutamate also inhibits wind elicited A4I1 responses. Although attempts were made to further characterize the receptor involved, tested substances such as kainic acid, glycine, CNQX or GDEE had no effect.     

神经元突触囊泡循环的分子机理

神经末梢突触囊泡循环包括锚靠、出胞、入胞及囊泡再生等步骤,由囊泡、轴浆及突触前膜的多种蛋白质的级联反应介导,其关键步骤的分子模型的确立,为进一步了解神经系统高级活动奠定了基础。     

Optical monitoring of synaptic vesicle trafficking in ribbon synapses

Synaptic transmission constitutes the major basis of communication among nerve cells. Upon nerve terminal depolarisation, calcium influx triggers the exocytosis of synaptic vesicles at active zones. Vesicles are then retrieved by endocytosis, recycled and refilled with neurotransmitter. Fluorescent styryl dyes have proven very useful as tools for studying several aspects of the synaptic vesicle cycle. Here, we review recent imaging studies using styryl FM dyes and bipolar cells of goldfish retina, which have a giant synaptic terminal containing ribbon-type active zones. Optical techniques applied to this unique synaptic terminal have provided novel insights into the trafficking of synaptic vesicles during and following strong stimulation.     

Identification of a novel process limiting the rate of synaptic vesicle cycling at hippocampal synapses

During intense presynaptic activity, the readily releasable pool (RRP) of synaptic vesicles empties more quickly than it can be refilled, and short-term depression results. Ordinarily, the pool refills within 20 s, but long, high-frequency trains of action potentials often induce a form of short-term depression that persists for a much longer time. Here, we report that replenishment of the RRP is governed by two simple processes: the previously identified mechanism termed refilling, and another process that appears after extensive exocytosis and produces a transient decrease in the capacity of the pool, lasting for several minutes. The data presented here place stringent constraints on the types of kinetic models that can be used to describe synaptic vesicular cycling and are inconsistent with the traditional multipool models of vesicular mobilization.     

The synaptic vesicle: cycle of exocytosis and endocytosis

Synaptic vesicles are clustered at the presynaptic terminal where they fuse and recycle in response to stimulation. Vesicles appear to be sorted into pools, but we do not yet understand how physiologically defined pools relate to morphological pools. The advent of dynamic imaging approaches has led to an appreciation of the regulation of vesicle mobility. Newly endocytosed vesicles are highly mobile but appear to become transiently trapped as they re-enter the recycling pool. Recent experiments indicate that endocytosis might have a constant rate, but limited capacity. How endocytosis is linked to exocytosis remains unclear, although calcium emerges as an important player.     

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.     

Ultrastructure of synapses in the central nervous system of lamellibranch molluscs.

Fine-structural characteristics of synaptic contacts were investigated in the central nervous system of different species of lamellibranch molluscs. Neuropile of the ganglia is characterized by regular occurrence of ultrastructurally well-defined polarized chemical synapses resembling those described in other invertebrate species and vertebrates. In addition to the generally observed membrane thickenings, enhanced density of synaptic membranes, cleft material and vesicle clustering on the presynaptic membrane, synapses are occasionally characterized by other and pinocytotic invaginations. Synaptic connections were distinguished on the basis of the vesicle content of the presynaptic terminal. Different forms of synaptic configurations (divergence, convergence, presynaptic modification) were observed in the ganglia.     

Fine-tuning the central nervous system: microglial modelling of cells and synapses

Microglia constitute as much as 10–15% of all cells in the mammalian central nervous system (CNS) and are the only glial cells that do not arise from the neuroectoderm. As the principal CNS immune cells, microglial cells represent the first line of defence in response to exogenous threats. Past studies have largely been dedicated to defining the complex immune functions of microglial cells. However, our understanding of the roles of microglia has expanded radically over the past years. It is now clear that microglia are critically involved in shaping neural circuits in both the developing and adult CNS, and in modulating synaptic transmission in the adult brain. Intriguingly, microglial cells appear to use the same sets of tools, including cytokine and chemokine release as well as phagocytosis, whether modulating neural function or mediating the brain''s innate immune responses. This review will discuss recent developments that have broadened our views of neuro-glial signalling to include the contribution of microglial cells.