LES COMPARTIMENTS D''ACETYLCHOLINE DE L''ORGANE ELECTRIQUE DE LA TORPILLE ET LEURS MODIFICATIONS PAR LA STIMULATION

Abstract— Changes in ‘free’ and ‘bound’ acetylcholine before and after stimulation have been investigated in vivo and in slices of electric organ of Torpedo marmorata incubated or superfused with physiological saline solutions. Spontaneous miniature end-plate potentials could be recorded and on electrical stimulation discharges of up to 30 V could be elicited. The electrical response fell off rapidly on repetitive stimulation. ‘Bound’ acetylcholine is that which relhains after the tissue has been homogenized since any ‘free’ acetylcholine is hydrolysed by the esterases when the tissue is disrupted. ‘Free’ acetylcholine can therefore be determined as the difference between the total acetylcholine found when the tissue is extracted with trichloroacetic acid and that which remains when the tissue is homogenized. Most of the ‘bound’ acetylcholine is present in synaptic vesicles. Stimulation of the tissue until the electrical response had fallen was accompanied by a drop in the level of ‘free’ acetylcholine. Lowered calcium and increased magnesium concentrations in the medium caused a decrease in the electrical response to stimulation and a decrease in the fall of ‘free’ acetylcholine. In other experiments, a decrease of both compartments was noticed at the end of the stimulation period. However the drop in ‘bound’ acetylcholine could also be elicited after the ‘free’ had fallen, by continuing the stimulation. When anticholinesterases were put in the medium, acetylcholine released on stimulation could be collected. On pre-incubation of the slice with [¹⁴C]choline, the acetylcholine stores became labelled. The specific radioactivity of the ‘free’ acetylcholine fluctuated on serial stimulations, whereas the specific radioactivity of the ‘bound’ acetylcholine remained stable under these experimental conditions. It is concluded that the ‘free’ compartment of acetylcholine is the most immediately available for release on stimulation.     

Characterization of a membrane protein from cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata

Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.     

Amino acids as transmitters of synaptic excitation in neocortical sensory processes.

Few synaptic transmitters are known to exist that are not represented in some region or another, or at some layer or other, in the cerebral cortex of mammalian brain. The more difficult job than mere identification of which substances are present, is that of the assignment of particular functional role(s) of such substances, and as well, of determining upon exactly which element(s) of the known synaptic circuitry of neocortex, such transmitters operate. Current wisdom subscribes to the view that the excitatory amino acids, most likely L-glutamate, and L-aspartate but perhaps also L-cysteate, L-homocysteate, L-cysteine sulfinate or even (although much less likely) the endogenous dipeptide substance, N-acetyl-L-aspartyl-L-glutamate, are the major excitatory synaptic transmitters of intracortical (associational) fibres, of corticofugal projections, and, as this article will attest, of thalamocortical inputs, as well. What particular limits, or restrictions, are imposed upon these generalizations, such as whether the data pertain only to primary sensory areas or follow some other yet to be determined rule, remains to be discovered in future experiments. This paper first presents an overview of the advances in understanding that have come about during the past few decades concerning the synaptic roles of amino acid transmitters. Next, an experimental section presents new evidence based on release studies and the microiontophoretic approach, which supports the view that the amino acids, glutamate and aspartate, interact with specific, pharmacologically identified subtypes of receptors in neocortex as transmitters of synaptic excitation released from thalamic afferent terminals.     

An antiserum specific for cholinergic synaptic vesicles from electric organ

Rabbit antisera to highly purified synaptic vesicles from the electric organ of Narcine brasiliensis, an electric ray, reveal a unique population of synaptic vesicle antigens in addition to a population shared with other electric organ membranes. Synaptic vesicle antigens were detected by binding successively rabbit antivesicle serum and radioactive goat anti-rabbit serum. To remove antibodies directed against antigens common to synaptic vesicles and other electric organ fractions, the antivesicle serum was extensively preadsorbed against an electric organ membrane fraction that was essentially free of synaptic vesicles. The adsorbed serum retained 40% of its ability to bind to synaptic vesicles, suggesting that about half of the antigenic determinants are unique. Vesicle antigens were quantified with a radioimmunoassay (RIA) that utilized precipitation of antibody-antigen complexes with Staphylococcus aureus cells. By this assay, the vesicles, detected by their acetylcholine (ACh) content and the antigens detected by the RIA, have the same buoyant density after isopycnic centrifugation of crude membrane fractions on sucrose and glycerol density gradients. The ratio of ACh to antigenicity was constant across the vesicle peaks and was close to that observed for vesicles purified to homogeneity. Even though the vesicles make up only approximately 0.5% of the material in the original homogenate, the ratio of acetylcholine to vesicle antigenicity could still be measured and also was indistinguishable from that of pure vesicles. We conclude that synaptic vesicles contain unique antigenic determinants not present to any measurable extent in other fractions of the electric organ. Consequently, it is possible to raise a synaptic vesicle- specific antiserum that allows vesicles to be detected and quantified. These findings are consistent with earlier immunohistochemical observations of specific antibody binding to motor nerve terminals.     

Physical studies of the interactions of acetylcholine chloride with membrane constituents

The binding of acetylcholine to pure lipids, and lipids, proteins and lipoproteins extracted from synaptic membranes, was investigated by monolayer and n.m.r. techniques. No specific binding of acetylcholine could be detected at the concentration used, although its muscarinic and nicotinic antagonists [atropine and (+)-tubocurarine respectively] could be shown to interact with the membrane components. It is concluded that the binding of the nicotinic and muscarinic antagonists of acetylcholine is not necessarily indicative of the existence of a specific acetylcholine receptor. Measurements of the displacement of (45)Ca(2+) from monolayers of phosphatidylserine by acetylcholine and the variation of electrophoretic mobility of phosphatidylserine particles with concentration of acetylcholine indicated that in these systems acetylcholine was acting as a counterion at the negatively charged lipid interface. But studies of the salting-in and salting-out of negatively charged lipid aggregates showed that acetylcholine and other quaternary ammonium compounds did not here behave simply as counterions. Electrostrictively hydrated cations such as Na(+) and K(+) were found to salt out, whereas hydrophobically hydrated cations such as acetylcholine salted in such aggregates. The possible role of the hydration of acetylcholine in synaptic transmission is discussed.     

外源乙酰胆碱在丝瓜卷须弯曲中起“神经递质”的作用

用乙酰胆碱（ＡＣｈ）或毒扁豆碱处理离体丝瓜卷须不仅能增强机械刺激引发的卷曲程度,甚至可以直接引发卷须的弯曲运动。用阿托品预处理卷须可以抑制由机械刺激引发的电化学波传递,减弱ＡＣｈ对卷须弯曲运动的效应;而Ｄ－管箭毒预处理卷须则无此效应。说明在丝瓜卷须中ＡＣｈ是通过毒蕈碱型受体传递电化波并引起原生质收缩和快速弯曲运动,这与动物神经－平滑肌突触传递兴奋的机制很相似。     

Characterization, by Size, Density, Osmotic Fragility, and Immunoaffinity, of Acetylcholine- and Vasoactive Intestinal Polypeptide-Containing Storage Particles from Myenteric Neurones of the Guinea-Pig

When cytoplasmic extracts of guinea-pig myenteric neurones are submitted to centrifugal density gradient fractionation in a zonal rotor acetylcholine is bimodally distributed in the gradient, in a peak (I) rich in synaptic vesicles of the classic type and in a denser peak (II/VI) rich in densecored vesicles and vasoactive intestinal polypeptide (VIP). The putative stable synaptic vesicle markers synaptophysin (p38), vesicular proteoglycan, and Mg2+-activated ATPase were also bimodally distributed, with a peak coincident with peak I and another, broader peak embracing peak II/VI, and neighbouring peaks of other neuropeptides resolved from peak II/VI by the density gradient separation procedure used. To establish whether the stable markers, acetylcholine and VIP in peak II/VI were present in one particle or several, attempts were made to separate them by particle-exclusion chromatography and differential osmotic fragility. These were unsuccessful, leading us to conclude that the storage particles in peak II/VI contain both neurotransmitters and all three putative stable synaptic vesicle markers. It is suggested that such particles are the counterparts, in cholinergic neurones of the myenteric plexus, of the dense-cored, enkephalin- and noradrenaline-containing vesicles of certain adrenergic neurones and, like the latter, may exist in a precursor-product relationship with the classic synaptic vesicles containing the small-molecular-mass transmitters and found in the same nerve terminals.     

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.     

Coexistence and corelease of cholinergic and peptidergic transmitters in frog sympathetic ganglia

Both acetylcholine (ACh) and a peptide that resembles luteinizing hormone-releasing hormone (LHRH) serve as transmitters in sympathetic ganglia of the bullfrog. Although ACh is contained and released from both preganglionic B fibers, which form synaptic contacts with only B cells in the ganglia, and preganglionic C fibers, which are in synaptic contact with C cells only, the LHRH-like peptide is contained and released exclusively from preganglionic C fibers. The same preganglionic C fibers appear to supply both ACh and the LHRH-like peptide because the thresholds for the cholinergic fast excitatory postsynaptic potential (EPSP) correlate well with the thresholds for the peptidergic late slow EPSP recorded in the same C cell. Further, anatomical studies showed that almost all nerve terminals on C cells contained the LHRH-like peptide. Some of these same terminals must also contain and release. ACh, mediating the cholinergic fast EPSPs with millisecond synaptic delays. Therefore at least some, if not all, terminals of preganglionic C fibers contain and release both cholinergic and peptidergic transmitters.     

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.     

Ultrastructural Changes Induced by 12-O-Tetradecanoylphorbol 13-Acetate in Pure Cholinergic Synaptosomes of Torpedo Electric Organ

We have studied the morphological changes induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment on pure cholinergic synaptosomes from Torpedo electric organ. These changes were studied by both ultrathin sections and freeze-fracture techniques. We found that after a treatment with TPA, a redistribution of synaptic vesicles inside the nerve endings and exocytotic images could be observed. Also, TPA, under conditions that induced the acetylcholine release, did not change the density of intramembrane particles at the synaptosomal protoplasmic hemimembrane leaflet. Similar results were found when calcium was not present in the extrasynaptosomal medium, and our results suggest that acetylcholine release induced by phorbol ester is probably mediated by exocytosis of synaptic vesicles.     

Agrin-like molecules at synaptic sites in normal, denervated, and damaged skeletal muscles

Several lines of evidence have led to the hypothesis that agrin, a protein extracted from the electric organ of Torpedo, is similar to the molecules in the synaptic cleft basal lamina at the neuromuscular junction that direct the formation of acetylcholine receptor and acetylcholinesterase aggregates on regenerating myofibers. One such finding is that monoclonal antibodies against agrin stain molecules concentrated in the synaptic cleft of neuromuscular junctions in rays. In the studies described here we made additional monoclonal antibodies against agrin and used them to extend our knowledge of agrin-like molecules at the neuromuscular junction. We found that anti-agrin antibodies intensely stained the synaptic cleft of frog and chicken as well as that of rays, that denervation of frog muscle resulted in a reduction in staining at the neuromuscular junction, and that the synaptic basal lamina in frog could be stained weeks after degeneration of all cellular components of the neuromuscular junction. We also describe anti-agrin staining in nonjunctional regions of muscle. We conclude the following: (a) agrin-like molecules are likely to be common to all vertebrate neuromuscular junctions; (b) the long-term maintenance of such molecules at the junction is nerve dependent; (c) the molecules are, indeed, a component of the synaptic basal lamina; and (d) they, like the molecules that direct the formation of receptor and esterase aggregates on regenerating myofibers, remain associated with the synaptic basal lamina after muscle damage.     

The isolation of pure cholinergic synaptic vesicles from the electric organs of elasmobranch fish of the family Torpedinidae

1. Zonal centrifuging permitted the separation, on the milligram scale and in a form largely free from contamination by soluble cytoplasmic protein or membrane fragments derived from other structures, of synaptic vesicles from the purely cholinergic terminals of the electric organ of Torpedo. Up to 100g of tissue could be processed in a single run. 2. As much as 46% of the bound acetylcholine from the original tissue preparation was recovered as a single peak of density equivalent to 0.38m-sucrose-0.21m-NaCl and with a concentration of up to 680nmol of acetylcholine/mg of protein. 3. The limiting concentration of acetylcholine in isolated vesicles when allowance had been made for non-vesicular protein appeared to be about 600nmol/mg of protein. 4. Vesicle counts by a ;bead-tagging' procedure indicated an acetylcholine content of about 360mumol/ml of vesicles; thus the vesicle protein content would be about 60% (w/v). 5. Calculations showed that the core of the vesicle, accounting for about 55% of the vesicle volume, could be largely filled with acetylcholine and protein.     

The modulatory action of superlow doses of mediators on the functional activity of the neuronal membrane]

It was shown that acetylcholine and gamma-aminobutyric acid at extremely low concentrations, which do not activate receptor-binding ionic channels, modulate the activity of NA-K-pump and Na:Ca exchange, the intracellular level of cAMP and chemosensitivity of the neuronal membrane. These data provide a new evidence in support for Koshtoiants's enzymochemical hypothesis in which synaptic transmitters are considered to be metabolic modulators for postsynaptic neurones.     

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.     

Agrin and Synaptic Laminin Are Required to Maintain Adult Neuromuscular Junctions

As synapses form and mature the synaptic partners produce organizing molecules that regulate each other’s differentiation and ensure precise apposition of pre- and post-synaptic specializations. At the skeletal neuromuscular junction (NMJ), these molecules include agrin, a nerve-derived organizer of postsynaptic differentiation, and synaptic laminins, muscle-derived organizers of presynaptic differentiation. Both become concentrated in the synaptic cleft as the NMJ develops and are retained in adulthood. Here, we used mutant mice to ask whether these organizers are also required for synaptic maintenance. Deletion of agrin from a subset of adult motor neurons resulted in the loss of acetylcholine receptors and other components of the postsynaptic apparatus and synaptic cleft. Nerve terminals also atrophied and eventually withdrew from muscle fibers. On the other hand, mice lacking the presynaptic organizer laminin-α4 retained most of the synaptic cleft components but exhibited synaptic alterations reminiscent of those observed in aged animals. Although we detected no marked decrease in laminin or agrin levels at aged NMJs, we observed alterations in the distribution and organization of these synaptic cleft components suggesting that such changes could contribute to age-related synaptic disassembly. Together, these results demonstrate that pre- and post-synaptic organizers actively function to maintain the structure and function of adult NMJs.     

Taste buds as peripheral chemosensory processors

Taste buds are peripheral chemosensory organs situated in the oral cavity. Each taste bud consists of a community of 50–100 cells that interact synaptically during gustatory stimulation. At least three distinct cell types are found in mammalian taste buds – Type I cells, Receptor (Type II) cells, and Presynaptic (Type III) cells. Type I cells appear to be glial-like cells. Receptor cells express G protein-coupled taste receptors for sweet, bitter, or umami compounds. Presynaptic cells transduce acid stimuli (sour taste). Cells that sense salt (NaCl) taste have not yet been confidently identified in terms of these cell types. During gustatory stimulation, taste bud cells secrete synaptic, autocrine, and paracrine transmitters. These transmitters include ATP, acetylcholine (ACh), serotonin (5-HT), norepinephrine (NE), and GABA. Glutamate is an efferent transmitter that stimulates Presynaptic cells to release 5-HT. This chapter discusses these transmitters, which cells release them, the postsynaptic targets for the transmitters, and how cell–cell communication shapes taste bud signaling via these transmitters.     

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.     

Classical Noncholinergic Neurotransmitters and the Vesicular Transport System for Acetylcholine

Abstract: The acetylcholine transporter exhibits such low affinity and specificity for acetylchoiine that it appeared possible it could fail to select against other neurotransmitters. Potential interactions of classical noncholinergic neurotransmitters with cholinergic synaptic vesicles purified from electric organ were studied. No active transport of [³H]serotonin, [³H]noradrenaline, or [³H]glutamate occurred. Serotonin, noradrenaline, and N -acetylaspartyl glutamate inhibited active transport of [³H]acetylcholine by the vesicles. Dopamine previously had been shown to inhibit transport. Glutamate and γ-aminobutyric acid were shown here not to inhibit active transport of [³H]-acetylcholine. Noradrenaline was competitive with respect to [³H]acetylcholine in this effect. Serotonin, noradrenaline, and dopamine inhibited binding of [³H]vesamicol to the vesicles, and dopamine was a competitive inhibitor of the binding of this allosteric ligand of the acetylcholine transporter. The results indicate that the acetylcholine transporter does not transport any other classical neurotransmitter, but serotonin, noradrenaline, and dopamine bind to the acetylcholine site.     

Modern Concepts of Cholinergic Neurotransmission at the Motor Synapse

Cholinergic synaptic contact between motor neuron and skeletal muscle fiber is perhaps one of the core objects for investigations of molecular mechanisms underlying the communication between neurons and innervated cells. In the studies conducted on this object in the past few decades, a large amount of experimental data was obtained that substantially complemented a traditional view on synaptic transmission. In particular, it was established that (i) acetylcholine is released from the nerve ending in both quantal and nonquantal ways; (ii) molecular mechanisms of the processes of the quantal acetylcholine release—spontaneous and evoked by electrical stimuli—have unique features and can be regulated independently; (iii) acetylcholine release from the nerve ending is accompanied by a release of a number of synaptically active molecules modulating the processes of secretion or reception of the main mediator; (iv) signal molecules affecting the process of cholinergic neurotransmission can be released not only from the nerve ending but also from glial cells and muscle fiber; (v) molecular mechanisms of the regulation of synaptic transmission are highly diverse and go beyond the alteration of the number of the released acetylcholine quanta. Thus, the neuromuscular junction shall be deemed currently as complicated and adaptive synapse characterized by a wide range of multiloop intercellular signaling pathways between presynaptic motor neuron ending, muscle fiber, and glial cells ensuring a high safety factor of synaptic transmission and the possibility of its fine tuning.