Synaptic Vesicle Exocytosis

Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for “soluble NSF-attachment protein receptor”) and SM (for “Sec1/Munc18-like”) proteins. During fusion, vesicular and target SNARE proteins assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap around assembling trans-SNARE complexes to catalyze membrane fusion. After fusion, SNARE complexes are dissociated by the ATPase NSF (for “N-ethylmaleimide sensitive factor”). Fusion-competent conformations of SNARE proteins are maintained by chaperone complexes composed of CSPα, Hsc70, and SGT, and by nonenzymatically acting synuclein chaperones; dysfunction of these chaperones results in neurodegeneration. The synaptic membrane-fusion machinery is controlled by synaptotagmin, and additionally regulated by a presynaptic protein matrix (the “active zone”) that includes Munc13 and RIM proteins as central components.Synaptic vesicles are uniform organelles of ∼40 nm diameter that constitute the central organelle for neurotransmitter release. Each presynaptic nerve terminal contains hundreds of synaptic vesicles that are filled with neurotransmitters. When an action potential depolarizes the presynaptic plasma membrane, Ca²⁺-channels open, and Ca²⁺ flows into the nerve terminal to trigger the exocytosis of synaptic vesicles, thereby releasing their neurotransmitters into the synaptic cleft (Fig. 1). Ca²⁺ triggers exocytosis by binding to synaptotagmin; after exocytosis, vesicles are re-endocytosed, recycled, and refilled with neurotransmitters. Recycling can occur by multiple parallel pathways, either by fast recycling via local reuse of vesicles (“kiss-and-run” and “kiss-and-stay”), or by slower recycling via an endosomal intermediate (Fig. 1).Open in a separate windowFigure 1.The synaptic vesicle cycle. A presynaptic nerve terminal is depicted schematically as it contacts a postsynaptic neuron. The synaptic vesicle cycle consists of exocytosis (red arrows) followed by endocytosis and recycling (yellow arrows). Synaptic vesicles (green circles) are filled with neurotransmitters (NT; red dots) by active transport (neurotransmitter uptake) fueled by an electrochemical gradient established by a proton pump that acidifies the vesicle interior (vesicle acidification; green background). In preparation to synaptic exocytosis, synaptic vesicles are docked at the active zone, and primed by an ATP-dependent process that renders the vesicles competent to respond to a Ca²⁺-signal. When an action potential depolarizes the presynaptic membrane, Ca²⁺-channels open, causing a local increase in intracellular Ca²⁺ at the active zone that triggers completion of the fusion reaction. Released neurotransmitters then bind to receptors associated with the postsynaptic density (PSD). After fusion pore opening, synaptic vesicles probably recycle via three alternative pathways: local refilling with neurotransmitters without undocking (“kiss-and-stay”), local recycling with undocking (“kiss-and-run”), and full recycling of vesicles with passage through an endosomal intermediate. (Adapted from Südhof 2004.)Due to their small size, synaptic vesicles contain a limited complement of proteins that have been described in detail (Südhof 2004; Takamori et al. 2006). Although the functions of several vesicle components remain to be identified, most vesicle components participate in one of three processes: neurotransmitter uptake and storage, vesicle exocytosis, and vesicle endocytosis and recycling. In addition, it is likely that at least some vesicle proteins are involved in the biogenesis of synaptic vesicles and the maintenance of their exquisite uniformity and stability, but little is known about how vesicles are made, and what determines their size.     

Caenorhabditis elegans wsp-1 Regulation of Synaptic Function at the Neuromuscular Junction

The Rho GTPase members and their effector proteins, such as the Wiskott-Aldrich syndrome protein (WASP), play critical roles in regulating actin dynamics that affect cell motility, endocytosis, cell division, and transport. It is well established that Caenorhabditis elegans wsp-1 plays an essential role in embryonic development. We were interested in the role of the C. elegans protein WSP-1 in the adult nematode. In this report, we show that a deletion mutant of wsp-1 exhibits a strong sensitivity to the neuromuscular inhibitor aldicarb. Transgenic rescue experiments demonstrated that neuronal expression of WSP-1 rescued this phenotype and that it required a functional WSP-1 Cdc42/Rac interactive binding domain. WSP-1-GFP fusion protein was found localized presynaptically, immediately adjacent to the synaptic protein RAB-3. Strong genetic interactions with wsp-1 and other genes involved in different stages of synaptic transmission were observed as the wsp-1(gm324) mutation suppresses the aldicarb resistance seen in unc-13(e51), unc-11(e47), and snt-1 (md290) mutants. These results provide genetic and pharmacological evidence that WSP-1 plays an essential role to stabilize the actin cytoskeleton at the neuronal active zone of the neuromuscular junction to restrain synaptic vesicle release.     

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.     

The Quantal Nature of Synaptic Transmission at the Neuromuscular Junction of a Spider

Spontaneous and evoked release of transmitter at neuromuscular junctions in three different leg muscles of a tarantula (Dugesiella hentzi) was investigated. In most cases the spontaneous miniature potentials were released independently, although bursts from single synaptic junctions occasionally occurred. In contrast to recent findings in other arthropod muscles, focal extracellular recording from junctional areas revealed that the evoked release of transmitter quanta followed Poisson's theorem at low quantal content synaptic junctions in arachnid muscles.     

Reduced Expression of the Vesicular Acetylcholine Transporter and Neurotransmitter Content Affects Synaptic Vesicle Distribution and Shape in Mouse Neuromuscular Junction

In vertebrates, nerve muscle communication is mediated by the release of the neurotransmitter acetylcholine packed inside synaptic vesicles by a specific vesicular acetylcholine transporter (VAChT). Here we used a mouse model (VAChT KD^HOM) with 70% reduction in the expression of VAChT to investigate the morphological and functional consequences of a decreased acetylcholine uptake and release in neuromuscular synapses. Upon hypertonic stimulation, VAChT KD^HOM mice presented a reduction in the amplitude and frequency of miniature endplate potentials, FM 1–43 staining intensity, total number of synaptic vesicles and altered distribution of vesicles within the synaptic terminal. In contrast, under electrical stimulation or no stimulation, VAChT KD^HOM neuromuscular junctions did not differ from WT on total number of vesicles but showed altered distribution. Additionally, motor nerve terminals in VAChT KD^HOM exhibited small and flattened synaptic vesicles similar to that observed in WT mice treated with vesamicol that blocks acetylcholine uptake. Based on these results, we propose that decreased VAChT levels affect synaptic vesicle biogenesis and distribution whereas a lower ACh content affects vesicles shape.     

Intrinsic Neuronal Determinants Locally Regulate Extrasynaptic and Synaptic Growth at the Adult Neuromuscular Junction

Long-term functional plasticity in the nervous system can involve structural changes in terminal arborization and synaptic connections. To determine whether the differential expression of intrinsic neuronal determinants affects structural plasticity, we produced and analyzed transgenic mice overexpressing the cytosolic proteins cortical cytoskeleton–associated protein 23 (CAP-23) and growth-associated protein 43 (GAP-43) in adult neurons.

Like GAP-43, CAP-23 was downregulated in mouse motor nerves and neuromuscular junctions during the second postnatal week and reexpressed during regeneration. In transgenic mice, the expression of either protein in adult motoneurons induced spontaneous and greatly potentiated stimulus-induced nerve sprouting at the neuromuscular junction. This sprouting had transgene-specific features, with CAP-23 inducing longer, but less numerous sprouts than GAP-43. Crossing of the transgenic mice led to dramatic potentiation of the sprout-inducing activities of GAP-43 and CAP-23, indicating that these related proteins have complementary and synergistic activities. In addition to ultraterminal sprouting, substantial growth of synaptic structures was induced. Experiments with pre- and postsynaptic toxins revealed that in the presence of GAP-43 or CAP-23, sprouting was stimulated by a mechanism that responds to reduced transmitter release and may be independent of postsynaptic activation.

These results demonstrate the importance of intrinsic determinants in structural plasticity and provide an experimental approach to study its role in nervous system function.

     

Kismet Positively Regulates Glutamate Receptor Localization and Synaptic Transmission at the Drosophila Neuromuscular Junction

The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse that is structurally and functionally similar to mammalian glutamatergic synapses. These synapses can, as a result of changes in activity, alter the strength of their connections via processes that require chromatin remodeling and changes in gene expression. The chromodomain helicase DNA binding (CHD) protein, Kismet (Kis), is expressed in both motor neuron nuclei and postsynaptic muscle nuclei of the Drosophila larvae. Here, we show that Kis is important for motor neuron synaptic morphology, the localization and clustering of postsynaptic glutamate receptors, larval motor behavior, and synaptic transmission. Our data suggest that Kis is part of the machinery that modulates the development and function of the NMJ. Kis is the homolog to human CHD7, which is mutated in CHARGE syndrome. Thus, our data suggest novel avenues of investigation for synaptic defects associated with CHARGE syndrome.     

Interactions between Presynaptic Calcium Channels and Proteins Implicated in Synaptic Vesicle Trafficking and Exocytosis

Monoclonal antibodies were generated by immunizing mice with chick brain synaptic membranes and screening for immunoprecipitation of solubilized conotoxin GVIA receptors (N-type calcium channels). Antibodies against two synaptic proteins (p35--syntaxin 1 and p58--synaptotagmin) were produced and used to purify and characterize a ternary complex containing N-type channels associated with these two proteins. These results provided the first evidence for a specific interaction between presynaptic calcium channels and SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. Immunoprecipitation experiments supported the conclusion that syntaxin 1/SNAP-25/VAMP/synaptotagmin I or II complexes associate with N-type, P/Q-type, but not L-type calcium channels from rat brain nerve terminals. Immunofluorescent confocal microscopy at the frog neuromuscular junction was consistent with the co-localization of syntaxin 1, SNAP-25, and calcium channels, all of which are predominantly expressed at active zones of the presynaptic plasma membrane facing post-synaptic folds rich in acetylcholine receptors. The interaction of proteins implicated in calcium-dependent exocytosis with presynaptic calcium channels may locate the sensor(s) that trigger vesicle fusion within a microdomain of calcium entry.     

Drosophila Neuroligin3 Regulates Neuromuscular Junction Development and Synaptic Differentiation

Neuroligins (Nlgs) are a family of cell adhesion molecules thought to be important for synapse maturation and function. Mammalian studies have shown that different Nlgs have different roles in synaptic maturation and function. In Drosophila melanogaster, the roles of Drosophila neuroligin1 (DNlg1), neuroligin2, and neuroligin4 have been examined. However, the roles of neuroligin3 (dnlg3) in synaptic development and function have not been determined. In this study, we used the Drosophila neuromuscular junctions (NMJs) as a model system to investigate the in vivo role of dnlg3. We showed that DNlg3 was expressed in both the CNS and NMJs where it was largely restricted to the postsynaptic site. We generated dnlg3 mutants and showed that these mutants exhibited an increased bouton number and reduced bouton size compared with the wild-type (WT) controls. Consistent with alterations in bouton properties, pre- and postsynaptic differentiations were affected in dnlg3 mutants. This included abnormal synaptic vesicle endocytosis, increased postsynaptic density length, and reduced GluRIIA recruitment. In addition to impaired synaptic development and differentiation, we found that synaptic transmission was reduced in dnlg3 mutants. Altogether, our data showed that DNlg3 was required for NMJ development, synaptic differentiation, and function.     

Botulinum Neurotoxin Type A is Internalized and Translocated from Small Synaptic Vesicles at the Neuromuscular Junction

Botulinum neurotoxin type A (BoNT/A) is the most frequent cause of human botulism and, at the same time, is largely used in human therapy. Some evidence indicates that it enters inside nerve terminals via endocytosis of synaptic vesicles, though this has not been directly proven. The metalloprotease L chain of the neurotoxin then reaches the cytosol in a process driven by low pH, but the acidic compartment wherefrom it translocates has not been identified. Using immunoelectron microscope, we show that BoNT/A does indeed enter inside synaptic vesicles and that each vesicle contains either one or two toxin molecules. This finding indicates that it is the BoNT/A protein receptor synaptic vesicle protein 2, and not its polysialoganglioside receptor that determines the number of toxin molecules taken up by a single vesicle. In addition, by rapid quenching the vesicle trans-membrane pH gradient, we show that the neurotoxin translocation into the cytosol is a fast process. Taken together, these results strongly indicate that translocation of BoNT/A takes place from synaptic vesicles, and not from endosomal compartments, and that the translocation machinery is operated by no more than two neurotoxin molecules.     

Mechanisms of the Effect of Dimephosphone on Synaptic Transmission in the Frog Neuromuscular Junction

We studied the influence of dimephosphone, an organophosphorus drug with a broad spectrum of therapeutic effects on the peripheral and central nervous systems, on postsynaptic end-plate currents (EPC) in the frog neuromuscular junction. Dimephosphone was demonstrated to decrease in a voltage-independent manner the EPC amplitude and to prolong the EPC decay. These effects are not related to inhibition of acetylcholinesterase. We propose a theoretical interpretation of the observed phenomena based on the model of blockade of an open ion channel of the acetylcholine receptor and conclude that postsynaptic receptors are one of the most probable targets for the action of dimephosphone.     

Acetylcholine Synthesis at the Rat Neuromuscular Junction

Acetylcholine (ACh) synthesis in homogenates of rat soleus muscles had two components. One component, specifically inhibited by bromoacetylcholine (BrACh), had a Km for choline of 0.26 mM; the other, resistant to BrACh, had a Km for choline of 45 mM. The component with a low Km was absent from denervated muscle, and was identical in kinetic terms to ACh synthesising activity in homogenates of sciatic nerve. It is therefore considered choline acetyltransferase (ChAT)-specific. The use of BrACh as a specific inhibitor of ChAT activity allowed the calculation of ACh synthesis at individual motor end-plates in the soleus muscle of the rat: 2.1 X 10(-3) nmol h-1. Since the number of muscle fibres and the number of motor units are known for this muscle, ACh synthesis per motor unit could be calculated: 0.15 nmol h-1. It is concluded that BrACh can be used as a specific inhibitor of ChAT activity in homogenates of skeletal muscle and that its use will obviate the necessity of dividing biopsied muscle or small rodent muscles into neural and aneural segments.     

Cholinergic Modulation of Acetylcholine Secretion at the Neuromuscular Junction

Journal of Evolutionary Biochemistry and Physiology - The effect of cholinergic compounds (activators and blockers of nicotinic cholinergic receptors) on acetylcholine secretion from motor nerve...     

Phenobarbital Increases Spontaneous Transmitter Release at the Frog Neuromuscular Junction

Phenobarbital (1-2 × 10^-4M) markedly increases the frequency of miniature end-plate potentials at the neuromuscular synapse of the frog. This effect was seen in calcium free media containing EGTA. The drug probably acts presynaptically at an intracellular locus to increase the presynaptic free calcium concentration.     

An Excess-Calcium-Binding-Site Model Predicts Neurotransmitter Release at the Neuromuscular Junction

Despite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important neural systems. Here, we report the development of a spatially realistic computational model of an entire frog active zone in which we constrained model parameters with experimental data, and then used Monte Carlo simulation methods to predict the Ca²⁺-binding stoichiometry and dynamics that underlie neurotransmitter release. Our model reveals that 20–40 independent Ca²⁺-binding sites on synaptic vesicles, only a fraction of which need to bind Ca²⁺ to trigger fusion, are sufficient to predict physiological release. Our excess-Ca²⁺-binding-site model has many functional advantages, agrees with recent data on synaptotagmin copy number, and is the first (to our knowledge) to link detailed physiological observations with the molecular machinery of Ca²⁺-triggered exocytosis. In addition, our model provides detailed microscopic insight into the underlying Ca²⁺ dynamics during synapse activation.     

An Excess-Calcium-Binding-Site Model Predicts Neurotransmitter Release at the Neuromuscular Junction

Despite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important neural systems. Here, we report the development of a spatially realistic computational model of an entire frog active zone in which we constrained model parameters with experimental data, and then used Monte Carlo simulation methods to predict the Ca²⁺-binding stoichiometry and dynamics that underlie neurotransmitter release. Our model reveals that 20–40 independent Ca²⁺-binding sites on synaptic vesicles, only a fraction of which need to bind Ca²⁺ to trigger fusion, are sufficient to predict physiological release. Our excess-Ca²⁺-binding-site model has many functional advantages, agrees with recent data on synaptotagmin copy number, and is the first (to our knowledge) to link detailed physiological observations with the molecular machinery of Ca²⁺-triggered exocytosis. In addition, our model provides detailed microscopic insight into the underlying Ca²⁺ dynamics during synapse activation.