scholarly journals EFFECTS OF CALCIUM-CONTAINING FIXATION SOLUTIONS ON CHOLINERGIC SYNAPTIC VESICLES

1974 ◽  
Vol 63 (3) ◽  
pp. 780-795 ◽  
Author(s):  
Alan F. Boyne ◽  
Timothy P. Bohan ◽  
Terence H. Williams
Keyword(s):  
Binding Sites ◽  
Synaptic Vesicles ◽  
Chelating Agents ◽  
Electric Organ ◽  
Morphological Evidence ◽  
Nerve Terminals ◽  
Vesicle Membrane ◽  
Dense Particle ◽  
Thin Sections ◽  
Electron Dense Particle

Calcium (Ca)-containing fixation solutions applied to slices of electric organ of the electric ray, Narcine brasiliensis, have been shown to have three distinct ultrastructural effects on cholinergic synaptic vesicles of the nerve terminals. (a) An electron-dense particle (EDS) is observed within the vesicle; the particle is seen in unosmicated, unstained tissues and can be removed from thin sections by Ca-chelating agents. It is concluded that the EDS represents Ca bound by the vesicle. It is suggested that the bound ATP of the vesicle provides anionic Ca binding sites. (b) The vesicle membrane tends to ‘crinkle’ or collapse depending on the concentration of the other components of the fixative solution. The ‘crinkling’ or collapse are largely reversed by a wash step in the absence of Ca. (c) The presence of Ca results in the appearance of a population of vesicles which form characteristic fusions or ‘tight’ junctions with the terminal membrane. This appears to be morphological evidence for the proposal, which has been frequently put forward, that Ca facilitates such a fusion before discharge of vesicle-bound transmitter. With the discovery that the use of Ca-containing fixatives leads to the demonstration of a subpopulation of synaptic vesicles fused to the terminal membrane, we are led to propose that this is the ultrastructural location of the newly synthesized acetylcholine which has been shown by others to be preferentially released by stimulation.

scholarly journals An antiserum specific for cholinergic synaptic vesicles from electric organ.

1980 ◽  
Vol 87 (1) ◽  
pp. 98-103 ◽  
Author(s):  
S S Carlson ◽  
R B Kelly
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Membrane Fraction ◽  
Motor Nerve ◽  
Buoyant Density ◽  
Electric Organ ◽  
Rabbit Serum ◽  
Antigenic Determinants ◽  
Nerve Terminals ◽  
Isopycnic Centrifugation

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.

scholarly journals Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells.

1985 ◽  
Vol 100 (4) ◽  
pp. 1284-1294 ◽  
Author(s):  
K Buckley ◽  
R B Kelly
Keyword(s):  
Synaptic Vesicles ◽  
Endocrine Cells ◽  
Electric Organ ◽  
Cell Types ◽  
Cross Reactivity ◽  
Secretory Vesicles ◽  
Vesicle Membrane ◽  
Electron Microscopic ◽  
Transmembrane Glycoprotein ◽  
Immunoperoxidase Labeling

Several types of cells store proteins in secretory vesicles from which they are released by an appropriate stimulus. It might be expected that the secretory vesicles in different cell types use similar molecular machinery. Here we describe a transmembrane glycoprotein (Mr approximately 100,000) that is present in secretory vesicles in all neurons and endocrine cells studied, in species from elasmobranch fish to mammals, and in neural and endocrine cell lines. It was detected by cross-reactivity with monoclonal antibodies raised to highly purified cholinergic synaptic vesicles from the electric organ of fish. By immunoprecipitation of intact synaptic vesicles and electron microscopic immunoperoxidase labeling, we have shown that the antigenic determinant is on the cytoplasmic face of the synaptic vesicles. However, the electrophoretic mobility of the antigen synthesized in the presence of tunicamycin is reduced to Mr approximately 62,000, which suggests that the antigen is glycosylated and must therefore span the vesicle membrane.

scholarly journals Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes).

1978 ◽  
Vol 78 (3) ◽  
pp. 685-700 ◽  
Author(s):  
R C Fried ◽  
M P Blaustein
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Plasma Membranes ◽  
Large Diameter ◽  
Small Diameter ◽  
Physiological Saline ◽  
Nerve Terminals ◽  
Vesicle Membrane ◽  
Thorium Dioxide ◽  
Vesicular Exocytosis

The morphological features of pinched-off presynaptic nerve terminals (synaptosomes) from rat brain were examined with electron microscope techniques; in many experiments, an extracellular marked (horseradish peroxidase or colloidal thorium dioxide) was included in the incubation media. When incubated in physiological saline, most terminals appeared approximately spherical, and were filled with small (approximately 400-A diameter) "synaptic vesicles"; mitochondria were also present in many of the terminals. In a number of instances the region of synaptic contact, with adhering portions of the postsynaptic cell membrane and postsynaptic density, could be readily discerned. Approximately 20--30% of the terminals in our preparations exhibited clear evidence of damage, as indicated by diffuse distribution of extracellular markers in the cytoplasm; the markers appeared to be excluded from the intraterminal vesicles under these circumstances. The markers were excluded from the cytoplasm in approximately 70--80% of the terminals, which may imply that these terminals have intact plasma membranes. When the terminals were treated with depolarizing agents (veratridine or K-rich media), in the presence of Ca, many new, large (600--900-A diameter) vesicles and some coated vesicles and new vacuoles appeared. When the media contained an extracellular marker, the newly formed structures frequently were labeled with the marker. If the veratridine-depolarized terminals were subsequently treated with tetrodotoxin (to repolarize the terminals) and allowed to "recover" for 60--90 min, most of the large marker-containing vesicles disappeared, and numerous small (approximately 400-A diameter) marker-containing vesicles appeared. These observations are consistent with the idea that pinched-off presynaptic terminals contain all of the machinery necessary for vesicular exocytosis and for the retrieval and recycling of synaptic vesicle membrane. The vesicle membrane appears to be retrieval primarily in the form of large diameter vesicles which are subsequently reprocessed to form new "typical" small-diameter synaptic vesicles.

scholarly journals EVIDENCE FOR RECYCLING OF SYNAPTIC VESICLE MEMBRANE DURING TRANSMITTER RELEASE AT THE FROG NEUROMUSCULAR JUNCTION

1973 ◽  
Vol 57 (2) ◽  
pp. 315-344 ◽  
Author(s):  
J. E. Heuser ◽  
T. S. Reese
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Motor Nerve ◽  
Coated Vesicles ◽  
Synaptic Membrane ◽  
Nerve Terminals ◽  
Frog Neuromuscular Junction ◽  
Vesicle Membrane ◽  
Intracellular Compartments

When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae.

scholarly journals Antibodies to synaptic vesicles purified from Narcine electric organ bind a subclass of mammalian nerve terminals.

1980 ◽  
Vol 87 (1) ◽  
pp. 104-113 ◽  
Author(s):  
J E Hooper ◽  
S S Carlson ◽  
R B Kelly
Keyword(s):  
Nervous System ◽  
Synaptic Vesicles ◽  
Chromaffin Cells ◽  
Molecular Layer ◽  
Electric Organ ◽  
Antigenic Determinants ◽  
Nerve Terminals ◽  
The Central Nervous System ◽  
Adrenal Chromaffin ◽  
Mammalian Nerve

Antibodies were raised in rabbits to synaptic vesicles purified to homogeneity from the electric organ of Narcine brasiliensis, a marine electric ray. These antibodies were shown by indirect immunofluorescence techniques to bind a wide variety of nerve terminals in the mammalian nervous system, both peripheral and central. The shared antigenic determinants are found in cholinergic terminals, including the neuromuscular junction, sympathetic ganglionic and parasympathetic postganglionic terminals, and in those synaptic areas of the hippocampus and cerebellum that stain with acetylcholinesterase. They are also found in some noncholinergic regions, including adrenergic sympathetic postganglionic terminals, the peptidergic terminals in the posterior pituitary, and adrenal chromaffin cells. They are, however, not found in many noncholinergic synapse-rich regions. Such regions include the molecular layer of the cerebellum and those laminae of the dentate gyrus that receive hippocampal associational and commissural input. We conclude that one or more of the relatively small number of antigenic determinants in pure electric fish synaptic vesicles have been conserved during evolution, and are found in some but not all nerve terminals of the mammalian nervous system. The pattern of antibody binding in the central nervous system suggests unexpected biochemical similarities between nerve terminals heretofore regarded as unrelated.

scholarly journals Redistribution of synaptophysin and synapsin I during alpha-latrotoxin-induced release of neurotransmitter at the neuromuscular junction.

1990 ◽  
Vol 110 (2) ◽  
pp. 449-459 ◽  
Author(s):  
F Torri-Tarelli ◽  
A Villa ◽  
F Valtorta ◽  
P De Camilli ◽  
P Greengard ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Low Dose ◽  
Synapsin I ◽  
Nerve Terminals ◽  
Frozen Sections ◽  
Vesicle Membrane ◽  
Ultrathin Frozen Sections ◽  
Nerve Muscle ◽  
Molecular Components

The distribution of two synaptic vesicle-specific phosphoproteins, synaptophysin and synapsin I, during intense quantal secretion was studied by applying an immunogold labeling technique to ultrathin frozen sections. In nerve-muscle preparations treated for 1 h with a low dose of alpha-latrotoxin in the absence of extracellular Ca2+ (a condition under which nerve terminals are depleted of both quanta of neurotransmitter and synaptic vesicles), the immunolabeling for both proteins was distributed along the axolemma. These findings indicate that, in the presence of a block of endocytosis, exocytosis leads to the permanent incorporation of the synaptic vesicle membrane into the axolemma and suggest that, under this condition, at least some of the synapsin I molecules remain associated with the vesicle membrane after fusion. When the same dose of alpha-latrotoxin was applied in the presence of extracellular Ca2+, the immunoreactivity patterns resembled those obtained in resting preparations: immunogold particles were selectively associated with the membrane of synaptic vesicles, whereas the axolemma was virtually unlabeled. Under this condition an active recycling of both quanta of neurotransmitter and vesicles operates. These findings indicate that the retrieval of components of the synaptic vesicle membrane is an efficient process that does not involve extensive intermixing between molecular components of the vesicle and plasma membrane, and show that synaptic vesicles that are rapidly recycling still have the bulk of synapsin I associated with their membrane.

Electron-dense Particle in Cholinergic Synaptic Vesicles

Nature ◽  
1973 ◽  
Vol 244 (5410) ◽  
pp. 32-34 ◽  
Author(s):  
T. P. BOHAN ◽  
A. F. BOYNE ◽  
P. S. GUTH ◽  
Y. NARAYANAN ◽  
T. H. WILLIAMS
Keyword(s):  
Synaptic Vesicles ◽  
Dense Particle ◽  
Electron Dense Particle

scholarly journals Membrane recycling in the cone cell endings of the turtle retina.

1978 ◽  
Vol 79 (3) ◽  
pp. 802-825 ◽  
Author(s):  
S F Schaeffer ◽  
E Raviola
Keyword(s):  
Synaptic Vesicle ◽  
Cold Exposure ◽  
Synaptic Vesicles ◽  
Surface Density ◽  
Room Temperature ◽  
Coated Vesicles ◽  
Vesicle Membrane ◽  
Cone Cell ◽  
Dark Light ◽  
Thin Sections

The ultrastructural effects of dark, light, and low temperature were investigated in the cone cell endings of the red-eared turtle (Pseudemys scripta elegans). Thin sections revealed that in dark-adapted retinas maintained at 22 degrees C, the neural processes which contact the cone cells at the invaginating synapses penetrated deeply into the photoreceptor endings. When dark-adapted retinas were illuminated for 1 h at 22 degrees C, the invaginating processes were apparently extruded from the synaptic endings. On the other hand, 1-h exposure to a temperature of 4 degrees C in the dark caused the invaginating processes to become much more strikingly inserted than at room temperature. A morphometric analysis showed that the ratio between the synaptic surface density of the endings and their total surface density decreased in the light and increased in the dark and cold. Freeze-fracturing documented fusion of synaptic vesicles with the presynaptic membrane in all conditions tested. These observations suggest that the changes in configuration of the pedicles in the light, dark, and cold reflect a different balance between addition and retrieval of synaptic vesicle membrane from the plasmalemma; in the dark, the rate of vesicle fusion is increased, whereas in the cold, membrane retrieval is blocked. When the eyecups were warmed up and illuminated for 30-45 min after cold exposure, a striking number of vacuoles and cisterns appeared in the cytoplasm and coated vesicles were commonly seen budding from the plasmalemma. 60-90 min after returning to room temperature, the endings had reverted to their normal configuration, and the vast majority of vacuoles, cisterns, and coated vesicles had disappeared. When horseradish peroxidase was included in the incubation medium, very few synaptic vesicles were labeled at the end of the period of cold exposure. 30-45 min after returning to 22 degrees C, vacuoles and cisterns contained peroxidase, whereas most synaptic vesicles were devoid of reaction product. 2 h after returning to 22 degrees C, coated vesicles, vacuoles, and cisterns had disappeared and a number of synaptic vesicles were labeled. These experiments suggest that vacuoles, cisterns, and coated vesicles mediate the retrieval of the synaptic vesicle membrane that has been added to the plasmalemma during cold exposure.

Empty Synaptic Vesicles Recycle and Undergo Exocytosis at Vesamicol-Treated Motor Nerve Terminals

1999 ◽  
Vol 81 (6) ◽  
pp. 2696-2700 ◽  
Author(s):  
Rodney L. Parsons ◽  
Michelle A. Calupca ◽  
Laura A. Merriam ◽  
Chris Prior
Keyword(s):  
Synaptic Vesicles ◽  
Motor Nerve ◽  
High Rate ◽  
Synaptic Membrane ◽  
Nerve Terminals ◽  
Vesicle Membrane ◽  
Motor Nerve Terminals ◽  
Prolonged Stimulation ◽  
Nerve Muscle ◽  
Activity Dependent

Empty synaptic vesicles recycle and undergo exocytosis at vesamicol-treated motor nerve terminals. We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve–muscle preparations were treated with 5 μM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s−1 in 35 KP but then declined to <1 s−1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1–43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1–43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals.

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