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.     

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

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 as point-like precipitates in the synaptic vesicles of resting (electrically nonstimulated) motor nerve terminals. When performed at low temperature, the same procedure revealed spot-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 revealed diffused 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.     

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.     

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".     

The ultrastructural ionic fixation technique localizing acetylcholinesterase activity, reveals simultaneously acetylcholine-like cation in the synaptic vesicles of the motor nerve terminals

A new cytochemical technique is proposed for side by side localization of acetylcholine and of acetylcholinesterase activity of motor end-plate at ultrastructural level. The technique is based on the simultaneous "ionic fixation" of vesicular acetylcholine and of histochemical copper thiocholine precipitate with phosphomolybdic acid: the molybdic heteropolyanion forms insoluble salts with these two quaternary ammonium cations, providing in situ "acetylcholine phosphomolybdate" and "copper thiocholine phosphomolybdate". Both of them are osmium resistant; the electron dense precipitates allow for a fine localization of acetylcholine and acetylcholinesterase activity at electron microscopic level.     

Trafficking of vesicular neurotransmitter transporters

Vesicular neurotransmitter transporters are required for the storage of all classical and amino acid neurotransmitters in secretory vesicles. Transporter expression can influence neurotransmitter storage and release, and trafficking targets the transporters to different types of secretory vesicles. Vesicular transporters traffic to synaptic vesicles (SVs) as well as large dense core vesicles and are recycled to SVs at the nerve terminal. Some of the intrinsic signals for these trafficking events have been defined and include a dileucine motif present in multiple transporter subtypes, an acidic cluster in the neural isoform of the vesicular monoamine transporter (VMAT) 2 and a polyproline motif in the vesicular glutamate transporter (VGLUT) 1. The sorting of VMAT2 and the vesicular acetylcholine transporter to secretory vesicles is regulated by phosphorylation. In addition, VGLUT1 uses alternative endocytic pathways for recycling back to SVs following exocytosis. Regulation of these sorting events has the potential to influence synaptic transmission and behavior.     

The "ins" and "outs" of the high-affinity choline transporter CHT1

Maintenance of acetylcholine (ACh) synthesis depends on the activity of the high-affinity choline transporter (CHT1), which is responsible for the reuptake of choline from the synaptic cleft into presynaptic neurons. In this review, we discuss the current understanding of mechanisms involved in the cellular trafficking of CHT1. CHT1 protein is mainly found in intracellular organelles, such as endosomal compartments and synaptic vesicles. The presence of CHT1 at the plasma membrane is limited by rapid endocytosis of the transporter in clathrin-coated pits in a mechanism dependent on a dileucine-like motif present in the carboxyl-terminal region of the transporter. The intracellular pool of CHT1 appears to constitute a reserve pool of transporters, important for maintenance of cholinergic neurotransmission. However, the physiological basis of the presence of CHT1 in intracellular organelles is not fully understood. Current knowledge about CHT1 indicates that stimulated and constitutive exocytosis, in addition to endocytosis, will have major consequences for regulating choline uptake. Future investigations of CHT1 trafficking should elucidate such regulatory mechanisms, which may aid in understanding the pathophysiology of diseases that affect cholinergic neurons, such as Alzheimer's disease.     

Silicotungstic acid for cytochemical localization of water soluble substance(s) of cholinergic motor nerve terminal

Silicotungstic acid (STA), an electron dense substance and a powerful precipitating agent of quaternary ammonium salts such as choline and acetylcholine, was employed on the frog motor end-plate in order to prove that STA reacts with diffusible substance(s) in nerve terminals. Thus, STA treatment and osmium tetroxide (OsO4) fixation were performed in three different ways. No reaction was detectable when STA treatment followed osmification, while simultaneous treatment with STA and OsO4 darkened both presynaptic and synaptic vesicle membranes. When STA was employed directly on fresh tissues which were subsequently fixed by OsO4, small black precipitates were observed in the synaptic vesicles and none on other synaptic structures. The possible reaction of STA with acetylcholine is discussed.     

A single packet of transmitter does not saturate postsynaptic glutamate receptors

Neurotransmitter is stored in synaptic vesicles and released by exocytosis into the synaptic cleft. One of the fundamental questions in central synaptic transmission is whether a quantal packet of transmitter saturates postsynaptic receptors. To address this question, we loaded the excitatory transmitter L-glutamate via whole-cell recording pipettes into the giant nerve terminal, the calyx of Held, in rat brainstem slices. This caused marked potentiations of both quantal and action potential-evoked EPSCs mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors. These results directly demonstrate that neither AMPA nor NMDA receptors are saturated by a single packet of transmitter, and indicate that vesicular transmitter content is an important determinant of synaptic efficacy.     

Transfection analysis of functional roles of complexin I and II in the exocytosis of two different types of secretory vesicles

Classical neurotransmitters such as gamma-aminobutyric acid and glutamate are released from synaptic nerve terminals by exocytosis of synaptic vesicles. PC12 cells also have SSVs capable of storing acetylcholine (ACh). A novel method to examine the effect of transient transfection of any gene of interest on the exocytosis of SSVs was developed. The transfection of choline acetyltransferase (ChAT) into PC12 cells which have lost ACh synthesizing activity resulted in the accumulation of a substantial amount of ACh. Synthesized ACh was released in Ca(2+)-dependent manner. Release was thought to occur by an exocytosis of SSVs because: (1) release was abolished by treating the cells with vesamicol, a specific inhibitor of the vesicular ACh transporter (VAChT) localizing specifically in SSVs; and (2) the release was further increased by cotransfecting rat VAChT with the ChAT. By means of this method, we showed that overexpression of complexin I or II with ChAT markedly suppressed high-K(+)-dependent ACh release of SSVs.     

Synaptopathy under conditions of altered gravity: Changes in synaptic vesicle fusion and glutamate release

Glutamate release and synaptic vesicle heterotypic/homotypic fusion were characterized in brain synaptosomes of rats exposed to hypergravity (10 G, 1 h). Stimulated vesicular exocytosis determined as KCl-evoked fluorescence spike of pH-sensitive dye acridine orange (AO) was decreased twice in synaptosomes under hypergravity conditions as compared to control. Sets of measurements demonstrated reduced ability of synaptic vesicles to accumulate AO (∼10% higher steady-state baseline level of AO fluorescence). Experiments with preloaded l-[¹⁴C]glutamate exhibited similar amount of total glutamate accumulated by synaptosomes, equal concentration of ambient glutamate, but the enlarged level of cytoplasmic glutamate measuring as leakage from digitonin-permeabilized synaptosomes in hypergravity. Thus, it may be suggested that +G-induced changes in stimulated vesicular exocytosis were a result of the redistribution of intracellular pool of glutamate, i.e. a decrease in glutamate content of synaptic vesicles and an enrichment of the cytoplasmic glutamate level. To investigate the effect of hypergravity on the last step of exocytosis, i.e. membrane fusion, a cell-free system consisted of synaptic vesicles, plasma membrane vesicles, cytosolic proteins isolated from rat brain synaptosomes was used. It was found that hypergravity reduced the fusion competence of synaptic vesicles and plasma membrane vesicles, whereas synaptosomal cytosolic proteins became more active to promote membrane fusion. The total rate of homo- and heterotypic fusion reaction initiated by Ca²⁺ or Mg²⁺/ATP remained unchanged under hypergravity conditions. Thus, hypergravity could induce synaptopathy that was associated with incomplete filling of synaptic vesicles with the neuromediator and changes in exocytotic release.     

Ectopic release of synaptic vesicles

Exocytosis of synaptic vesicles is generally assumed to occur only at ultrastructurally defined presynaptic active zones. If release is restricted to these sites, receptors not located within the synaptic cleft must be activated by transmitter that diffuses out of the cleft or not be activated at all. Here we report that AMPA receptor-mediated quantal events resulting from climbing fiber release are observed in Bergmann glial cells in the cerebellar cortex. These quantal events are not coincident with quanta recorded in neighboring Purkinje cells which receive input from the same climbing fiber. As Bergmann glial membranes are excluded from the synaptic cleft, we propose that exocytosis can occur from climbing fiber release sites located directly across from Bergmann glial membranes. Such ectopic release may account for the majority of the Bergmann glial AMPA response evoked by climbing fiber stimulation.     

Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells

Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH- terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs.     

Silicotungstic acid for cytochemical localization of water soluble substance(s) of cholinergic motor nerve terminal

Summary Silicotungstic acid (STA), an electron dense substance and a powerful precipitating agent of quaternary ammonium salts such as choline and acetylcholine, was employed on the frog motor end-plate in order to prove that STA reacts with diffusible substance(s) in nerve terminals. Thus, STA treatment and osmium tetroxide (OsO₄) fixation were performed in three different ways. No reaction was detectable when STA treatment followed osmification, while simultaneous treatment with STA and OsO₄ darkened both presynaptic and synaptic vesicle membranes. When STA was employed directly on fresh tissues which were subsequently fixed by OsO₄, small black precipitates were observed in the synaptic vesicles and none on other synaptic structures. The possible reaction of STA with acetylcholine is discussed.     

Exchangeability of radioactive acetylcholine with the bound acetylcholine of synaptosomes and synaptic vesicles

1. The exchangeability with added radioactive acetylcholine of the acetylcholine in isolated presynaptic nerve terminals (synaptosomes) and isolated synaptic vesicles was studied by a Sephadex-column method. 2. A substantial proportion of the synaptosomal acetylcholine is exchangeable with added radioactive acetylcholine. It is liberated by hypo-osmotic shock and ultrasonic treatment, and behaves as though it occupies the cytoplasmic compartment of synaptosomes. 3. Methods of isolating vesicles from hypo-osmotically ruptured synaptosomes in optimum yield are discussed. 4. The acetylcholine of synaptic vesicles isolated on a sucrose density gradient is released by hypo-osmotic conditions, suggesting that it is enclosed by a semi-permeable membrane; however, it is not easily released by ultrasonic treatment. 5. Added radioactive acetylcholine does not exchange with vesicular acetylcholine under a variety of different conditions. These include addition of ATP and Mg(2+), and pre-loading of the synaptosome with radioactive acetylcholine before hypo-osmotic rupture. This failure to exchange is discussed in terms of the possible storage mechanism of vesicular acetylcholine.     

Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).     

The loading of neurotransmitters into synaptic vesicles

Classical (non-peptide) transmitters are stored into secretory vesicles by a secondary active transporter driven by a V-type H(+)-ATPase. Five vesicular neurotransmitter uptake activities have been characterized in vitro and, for three of them, the transporters involved have been identified at the molecular level using cDNA cloning and/or Caenorhabditis elegans genetics. These transporters belong to two protein families, which are both unrelated to the Na(+)-coupled neurotransmitter transporters operating at the plasma membrane. The two isoforms of the mammalian vesicular monoamine transporter, VMAT1 and VMAT2, are related to the vesicular acetylcholine transporter (VACHT), while a novel, unrelated vesicular inhibitory amino acid transporter (VIAAT), also designated vesicular GABA transporter (VGAT), is responsible for the storage of GABA, glycine or, at some synapses, both amino acids into synaptic vesicles. The observed effects of experimentally altered levels of VACHT or VMAT2 on synaptic transmission and behavior, as well as the recent awareness that GABAergic or glutamatergic receptors are not always saturated at central synapses, suggest a potential role of vesicular loading in synaptic plasticity.     

Asymmetry of lipid organization in cholinergic synaptic vesicle membranes.

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.     

Recycled Synaptic Vesicles Contain Vesicle but Not Plasma Membrane Marker, Newly Synthesized Acetylcholine, and a Sample of Extracellular Medium

To monitor the fate of the synaptic vesicle membrane compartment, synaptic vesicles were isolated under varying experimental conditions from blocks of perfused Torpedo electric organ. In accordance with previous results, after low-frequency stimulation (0.1 Hz, 1,800 pulses) of perfused blocks of electric organ, a population of vesicles (VP2 type) can be separated by density gradient centrifugation and chromatography on porous glass beads that is denser and smaller than resting vesicles (VP1 type). By simultaneous application of fluorescein isothiocyanate-dextran as extracellular volume marker and [3H]acetate as precursor of vesicular acetylcholine, and by identifying the vesicular membrane compartment with an antibody against the synaptic vesicle transmembrane glycoprotein SV2, we can show that the membrane compartment of part of the synaptic vesicles becomes recycled during the stimulation period. It then contains both newly synthesized acetylcholine and a sample of extracellular medium. Recycled vesicles have not incorporated the presynaptic plasma membrane marker acetylcholinesterase. Cisternae or vacuoles are presumably not involved in vesicle recycling. After a subsequent period of recovery (18 h), all vesicular membrane compartments behave like VP1 vesicles on subcellular fractionation and still retain both volume markers. Our results imply that on low-frequency stimulation, synaptic vesicles are directly recycled, equilibrating their luminal contents with the extracellular medium and retaining their membrane identity and capability to accumulate acetylcholine.     

Neurotransmitter release at rapid synapses

The classical concept of the vesicular hypothesis for acetylcholine (ACh) release, one quantum resulting from exocytosis of one vesicle, is becoming more complicated than initially thought. 1) synaptic vesicles do contain ACh, but the cytoplasmic pool of ACh is the first to be used and renewed on stimulation. 2) The vesicles store not only ACh, but also ATP and Ca(2+) and they are critically involved in determining the local Ca(2+) microdomains which trigger and control release. 3) The number of exocytosis pits does increase in the membrane upon nerve stimulation, but in most cases exocytosis happens after the precise time of release, while it is a change affecting intramembrane particles which reflects more faithfully the release kinetics. 4) The SNARE proteins, which dock vesicles close to Ca(2+) channels, are essential for the excitation-release coupling, but quantal release persists when the SNAREs are inactivated or absent. 5) The quantum size is identical at the neuromuscular and nerve-electroplaque junctions, but the volume of a synaptic vesicle is eight times larger in electric organ; at this synapse there is enough ACh in a single vesicle to generate 15-25 large quanta, or 150-200 subquanta. These contradictions may be only apparent and can be resolved if one takes into account that an integral plasmalemmal protein can support the formation of ACh quanta. Such a protein has been isolated, characterised and called mediatophore. Mediatophore has been localised at the active zones of presynaptic nerve terminals. It is able to release ACh with the expected Ca(2+)-dependency and quantal character, as demonstrated using mediatophore-transfected cells and other reconstituted systems. Mediatophore is believed to work like a pore protein, the regulation of which is in turn likely to depend on the SNARE-vesicle docking apparatus.