Ultracytochemical localization of acetylcholine-like cations in excited motor end-plates by means of ionic fixation

Summary Using rapid ionic fixation with molybdic or tungstic heteropolyanions (strong precipitating agents of quaternary ammonium cations such as choline and acetylcholine), acetylcholine-like cations were localized aspoint-like precipitates in the synaptic vesicles of resting (electrically nonstimulated) motor nerve terminals. When performed at low temperature, the same procedure revealedspot-like precipitates (presumed to be exocytotically released acetylcholine-like cations) in the synaptic cleft in the vicinity of the active zone. These precipitates were often seen in paired forms. Unlike resting motor-nerve terminals, excited terminals (electrical stimulation with occasional 4-aminopyridine pretreatment) after ionic fixation exhibited, at first,laminar precipitates both in the vicinity of the active zone inside the nerve terminals and in the synaptic space. In the vicinity of the active zone, the laminar precipitates were directed towards the synaptic membrane, while those in the synaptic space showed no orientation. Ionic fixation also revealeddiffused precipitates both around the synaptic vesicles and on the axoplasmic side of the presynaptic membrane. Finally, the same fixation procedure demonstrated the presence of empty synaptic vesicles (without point-like precipitates) in close contact with the presynaptic membrane. The laminar and diffused precipitates are presumed to be two different forms of the same salts of acetylcholine-like cations that are insolubilized by ionic fixation in both the nerve terminals and the synaptic space of excited motor end-plates.     

Electron-microscope cytochemistry of acetylcholine-like cation by means of low-temperature "ionic fixation"

A fresh preparation of frog neuromuscle was fixed at low temperatures (0 degree-4 degrees C) by means of an "ionic-fixation" procedure which is based on the precipitation of quaternary ammonium cations, such as choline and acetylcholine, with molybdic or tungstic heteropolyanions. A low temperature was used to slow down drastically the biological processus of vesicular exocytosis so that ionic fixation, the speed of which is only slightly influenced by temperature variation, could be performed efficiently. In addition to the conventional point-like precipitate in the synaptic vesicle which is considered to be vesicular acetylcholine, numerous spot-like precipitates were observed in the synaptic cleft. Most of these were contiguous to the active zone, and some were in a paired form and corresponded to the double rows of the synaptic vesicles in contact with active zones. It is concluded that these spot-like precipitates were acetylcholine-like cations of the synaptic vesicles which had been discharged into the synaptic cleft by exocytosis and captured by the ionic fixation procedure. The results are discussed in relation to the vesicular and non-vesicular hypothesis of acetylcholine release.     

Electron-microscope cytochemistry of acetylcholine-like cation by means of low-temperature “ionic fixation”

Summary A fresh preparation of frog neuromuscle was fixed at low temperatures (0°–4°C) by means of an ionic-fixation procedure which is based on the precipitation of quaternary ammonium cations, such as choline and acetylcholine, with molybdic or tungstic heteropolyanions. A low temperature was used to slow down drastically the biological processus of vesicular exocytosis so that ionic fixation, the speed of which is only slightly influenced by temperature variation, could be performed efficiently. In addition to the conventional point-like precipitate in the synaptic vesicle which is considered to be vesicular acetylcholine, numerous spot-like precipitates were observed in the synaptic cleft. Most of these were contiguous to the active zone, and some were in a paired form and corresponded to the double rows of the synaptic vesicles in contact with active zones. It is concluded that these spot-like precipitates were acetylcholine-like cations of the synaptic vesicles which had been discharged into the synaptic cleft by exocytosis and captured by the ionic fixation procedure. The results are discussed in relation to the vesicular and non-vesicular hypothesis of acetylcholine release.     

Molybdic and tungstic heteropolyanions for "ionic fixation" of acetylcholine in cholinergic motor nerve terminals

The treatment of neuromuscular junctions with phosphomolybdic acid (PMA) and silicotungstic acid (STA) heteropolyanions permits the visualization of electron dense precipitates in the synaptic vesicles of the cholinergic motor nerve terminals. At the light microscopic level, the uncolored molybdenum salt is visualized after reduction to molybdenum blue. The blue coloration is confined to the nerve terminals. Since PMA and STA are known as strong precipitating agents of quaternary ammonium compounds (cations) it is supposed that they have insolubilized in situ the acetylcholine (Ach) of the synaptic vesicles by means of a rapid ionic interaction. Furthermore, in spite of the strong acidity of PMA and STA solutions, the ultrastructure of the treated tissue is not significantly altered but on the contrary seems to be well preserved. The ionic insolubilization of Ach, added to the good preservation of the ultrastructure prompted us to use the term "ionic fixation".     

Regulation of synaptic vesicle docking by different classes of macromolecules in active zone material

The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10-15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles' distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry.     

Unwebbing the presynaptic web

The release of neurotransmitter from nerve terminals occurs at a specialized region of the presynaptic plasma membrane called the active zone. A dense matrix of proteins associated with the active zone, called the presynaptic web, is thought to play a fundamental role in defining these neurotransmitter release sites. In this issue of Neuron, Phillips et al. have identified conditions for the biochemical purification of the presynaptic web and show that the web is comprised of proteins involved in the docking, fusion, and recycling of synaptic vesicles.     

The actin cytoskeleton and neurotransmitter release: an overview

Here we review evidence that actin and its binding partners are involved in the release of neurotransmitters at synapses. The spatial and temporal characteristics of neurotransmitter release are determined by the distribution of synaptic vesicles at the active zones, presynaptic sites of secretion. Synaptic vesicles accumulate near active zones in a readily releasable pool that is docked at the plasma membrane and ready to fuse in response to calcium entry and a secondary, reserve pool that is in the interior of the presynaptic terminal. A network of actin filaments associated with synaptic vesicles might play an important role in maintaining synaptic vesicles within the reserve pool. Actin and myosin also have been implicated in the translocation of vesicles from the reserve pool to the presynaptic plasma membrane. Refilling of the readily releasable vesicle pool during intense stimulation of neurotransmitter release also implicates synapsins as reversible links between synaptic vesicles and actin filaments. The diversity of actin binding partners in nerve terminals suggests that actin might have presynaptic functions beyond synaptic vesicle tethering or movement. Because most of these actin-binding proteins are regulated by calcium, actin might be a pivotal participant in calcium signaling inside presynaptic nerve terminals. However, there is no evidence that actin participates in fusion of synaptic vesicles.     

Molecular organization of the presynaptic active zone

The exocytosis of neurotransmitter-filled synaptic vesicles is under tight temporal and spatial control in presynaptic nerve terminals. The fusion of synaptic vesicles is restricted to a specialized area of the presynaptic plasma membrane: the active zone. The protein network that constitutes the cytomatrix at the active zone (CAZ) is involved in the organization of docking and priming of synaptic vesicles and in mediating use-dependent changes in release during short-term and long-term synaptic plasticity. To date, five protein families whose members are highly enriched at active zones (Munc13s, RIMs, ELKS proteins, Piccolo and Bassoon, and the liprins-α), have been characterized. These multidomain proteins are instrumental for the diverse functions performed by the presynaptic active zone.In our laboratories, work on the molecular organization of the active zone is supported by the Deutsche Forschungsgemeinschaft (Emmy Noether Fellowship, SFB645/A4 to S.S., SFB426/A1 to E.D.G.), the European Commission (SynScaff Consortium), the Land Sachsen-Anhalt (LSA-N2), the Fonds der Chemischen Industrie, and a Max Planck Research Award by the Max Planck Society, the Alexander von Humboldt Society, and local funding (BONFOR to S.S.).     

Neural Organization of the Median Ocellus of the Dragonfly : II. Synaptic structure

Two types of presumed synaptic contacts have been recognized by electron microscopy in the synaptic plexus of the median ocellus of the dragonfly. The first type is characterized by an electron-opaque, button-like organelle in the presynaptic cytoplasm, surrounded by a cluster of synaptic vesicles. Two postsynaptic elements are associated with these junctions, which we have termed button synapses. The second synaptic type is characterized by a dense cluster of synaptic vesicles adjacent to the presumed presynaptic membrane. One postsynaptic element is observed at these junctions. The overwhelming majority of synapses seen in the plexus are button synapses. They are found most commonly in the receptor cell axons where they synaptically contact ocellar nerve dendrites and adjacent receptor cell axons. Button synapses are also seen in the ocellar nerve dendrites where they appear to make synapses back onto receptor axon terminals as well as onto adjacent ocellar nerve dendrites. Reciprocal and serial synaptic arrangements between receptor cell axon terminals, and between receptor cell axon terminals and ocellar nerve dendrites are occasionally seen. It is suggested that the lateral and feedback synapses in the median ocellus of the dragonfly play a role in enhancing transients in the postsynaptic responses.     

The structure and function of ‘active zone material’ at synapses

The docking of synaptic vesicles on the presynaptic membrane and their priming for fusion with it to mediate synaptic transmission of nerve impulses typically occur at structurally specialized regions on the membrane called active zones. Stable components of active zones include aggregates of macromolecules, ‘active zone material’ (AZM), attached to the presynaptic membrane, and aggregates of Ca²⁺-channels in the membrane, through which Ca²⁺ enters the cytosol to trigger impulse-evoked vesicle fusion with the presynaptic membrane by interacting with Ca²⁺-sensors on the vesicles. This laboratory has used electron tomography to study, at macromolecular spatial resolution, the structure and function of AZM at the simply arranged active zones of axon terminals at frog neuromuscular junctions. The results support the conclusion that AZM directs the docking and priming of synaptic vesicles and essential positioning of Ca²⁺-channels relative to the vesicles'' Ca²⁺-sensors. Here we review the findings and comment on their applicability to understanding mechanisms of docking, priming and Ca²⁺-triggering at other synapses, where the arrangement of active zone components differs.     

Rab2 regulates presynaptic precursor vesicle biogenesis at the trans-Golgi

Reliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals, is prerequisite for successful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. We show here that in mutants of the small GTPase Rab2, both active zone and synaptic vesicle proteins accumulated in the neuronal cell body at the trans-Golgi and were, consequently, depleted at synaptic terminals, provoking neurotransmission deficits. Ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40 × 60 nm, segregating in subfractions either positive for active zone or synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, Rab2 acts upstream of Arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we identified a Golgi-associated assembly sequence of presynaptic precursor biogenesis dependent on a Rab2-regulated protein export and sorting step at the trans-Golgi.     

Membrane association of presynaptic cytomatrix protein bassoon

Components of the specialized cytomatrix at active zones of presynaptic nerve terminals are thought to be involved in organizing synaptic events such as immobilisation or translocation of synaptic vesicles and assemblingactive zone components. The 420-kDa non-transmembraneprotein Bassoon is a specific componentof the presynaptic cytomatrix that shares features with both cytoskeleton-associated and peripheral-membrane proteins. Using immunogold electron microscopy we show here that synapse associated Bassoon is distributed in a subregion of active zones. Using a biochemical assay we show that a fraction of Bassoon is membrane associated. Electron microscopy performed on the same biochemical fraction further revealed that Bassoon is associated with vesicular structures. Together these data suggest that at least a fraction of Bassoon is associated with a membraneous compartment in neurons.     

Some features of the submicroscopic morphology of synapses in frog and earthworm

Electron micrographs are presented of synaptic regions encountered in sections of frog sympathetic ganglia and earthworm nerve cord neuropile. Pre- and postsynaptic neuronal elements each appear to have a membrane 70 to 100 A thick, separated from each other over the synaptic area by an intermembranal space 100 to 150 A across. A granular or vesicular component, here designated the synaptic vesicles, is encountered on the presynaptic side of the synapse and consists of numerous oval or spherical bodies 200 to 500 A in diameter, with dense circumferences and lighter centers. Synaptic vesicles are encountered in close relationship to the synaptic membrane. In the earthworm neuropile elongated vesicles are found extending through perforations or gaps in the presynaptic membrane, with portions of vesicles appearing in the intermembranal space. Mitochondria are encountered in the vicinity of the synapse, and in the frog, a submicroscopic filamentary component can be seen in the presynaptic member extending up to the region where the vesicles are found, but terminating short of the synapse itself.     

SOME FEATURES OF THE SUBMICROSCOPIC MORPHOLOGY OF SYNAPSES IN FROG AND EARTHWORM

Electron micrographs are presented of synaptic regions encountered in sections of frog sympathetic ganglia and earthworm nerve cord neuropile. Pre- and postsynaptic neuronal elements each appear to have a membrane 70 to 100 A thick, separated from each other over the synaptic area by an intermembranal space 100 to 150 A across. A granular or vesicular component, here designated the synaptic vesicles, is encountered on the presynaptic side of the synapse and consists of numerous oval or spherical bodies 200 to 500 A in diameter, with dense circumferences and lighter centers. Synaptic vesicles are encountered in close relationship to the synaptic membrane. In the earthworm neuropile elongated vesicles are found extending through perforations or gaps in the presynaptic membrane, with portions of vesicles appearing in the intermembranal space. Mitochondria are encountered in the vicinity of the synapse, and in the frog, a submicroscopic filamentary component can be seen in the presynaptic member extending up to the region where the vesicles are found, but terminating short of the synapse itself.     

Rab3a is involved in transport of synaptic vesicles to the active zone in mouse brain nerve terminals

The rab family of GTP-binding proteins regulates membrane transport between intracellular compartments. The major rab protein in brain, rab3A, associates with synaptic vesicles. However, rab3A was shown to regulate the fusion probability of synaptic vesicles, rather than their transport and docking. We tested whether rab3A has a transport function by analyzing synaptic vesicle distribution and exocytosis in rab3A null-mutant mice. Rab3A deletion did not affect the number of vesicles and their distribution in resting nerve terminals. The secretion response upon a single depolarization was also unaffected. In normal mice, a depolarization pulse in the presence of Ca(2+) induces an accumulation of vesicles close to and docked at the active zone (recruitment). Rab3A deletion completely abolished this activity-dependent recruitment, without affecting the total number of vesicles. Concomitantly, the secretion response in the rab3A-deficient terminals recovered slowly and incompletely after exhaustive stimulation, and the replenishment of docked vesicles after exhaustive stimulation was also impaired in the absence of rab3A. These data indicate that rab3A has a function upstream of vesicle fusion in the activity-dependent transport of synaptic vesicles to and their docking at the active zone.     

The organization of cytoplasm at the presynaptic active zone of a central nervous system synapse

The axoplasm at the presynaptic active zone of excitatory synapses between parallel fibers and Purkinje cell spines contains a meshwork of distinct filaments intermingled with synaptic vesicles, seen most clearly after the rapid freezing, freeze-etch technique of tissue preparation. One set of filaments extends radially from synaptic vesicles and intersects similar filaments associated with vesicles as well as larger filaments arising from the presynaptic membrane. The small, vesicle-associated filaments appear to link synaptic vesicles to one another and to enmesh them in the vicinity of the synaptic junction. The vesicle-associated filaments could be synapsin I because they have the same molecular dimensions and are distributed in the same pattern as synapsin I immunoreactivity.     

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.     

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.     

Structure, isolation, composition and reconstitution of the neuronal fusion pore

Neuronal communication is dependent on the fusion of 40-50 nm in diameter synaptic vesicles containing neurotransmitters, at the presynaptic membrane. Here we report for the first time at 5-8A resolution, the presence of 8-10 nm in diameter cup-shaped neuronal fusion pores or porosomes at the presynaptic membrane, where synaptic vesicles dock and fuse to release neurotransmitters. The structure, isolation, composition, and functional reconstitution of porosomes present at the nerve terminal are described. These findings reveal the molecular mechanism of neurotransmitter release at the presynaptic membrane of nerve terminals.     

Intracellular movements of fluorescently labeled synaptic vesicles in frog motor nerve terminals during nerve stimulation.

We stained synaptic vesicles in frog motor nerve terminals with FM1-43 and studied changes in the shape and position of vesicle clusters during nerve stimulation. Each stained vesicle cluster appeared as a fluorescent spot. During repetitive nerve stimulation the spots gradually dimmed, most without changing shape or position. Occasionally, however, a spot moved, appearing in some cases to stream toward and coalesce with a neighboring spot. This suggests the existence of translocation mechanisms that can actively move vesicles in a coordinated fashion between vesicle clusters. Within single clusters, we saw no signs of such directed vesicle movements. Fluorescent spots in terminals viewed from the side with a confocal microscope did not shrink toward the presynaptic membrane during nerve stimulation, but dimmed uniformly. This suggests that vesicles continuously mix within a cluster during destaining and provides no evidence of active vesicle translocators within single vesicle clusters for moving vesicles to the presynaptic membrane.