scholarly journals Structural changes after transmitter release at the frog neuromuscular junction.

1981 ◽  
Vol 88 (3) ◽  
pp. 564-580 ◽  
Author(s):  
J E Heuser ◽  
T S Reese
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Structural Changes ◽  
Frog Neuromuscular Junction ◽  
Vesicle Membrane ◽  
Intramembrane Particles ◽  
Synaptic Vesicle Exocytosis ◽  
Quick Freezing ◽  
Vesicle Exocytosis ◽  
Active Zones

The sequence of structural changes that occur during synaptic vesicle exocytosis was studied by quick-freezing muscles at different intervals after stimulating their nerves, in the presence of 4-aminopyridine to increase the number of transmitter quanta released by each stimulus. Vesicle openings began to appear at the active zones of the intramuscular nerves within 3-4 ms after a single stimulus. The concentration of these openings peaked at 5-6 ms, and then declined to zero 50-100 ms late. At the later times, vesicle openings tended to be larger. Left behind at the active zones, after the vesicle openings disappeared, were clusters of large intramembrane particles. The larger particles in these clusters were the same size as intramembrane particles in undischarged vesicles, and were slightly larger than the particles which form the rows delineating active zones. Because previous tracer work had shown that new vesicles do not pinch off from the plasma membrane at these early times, we concluded that the particle clusters originate from membranes of discharged vesicles which collapse into the plasmalemma after exocytosis. The rate of vesicle collapse appeared to be variable because different stages occurred simultaneously at most times after stimulation; this asynchrony was taken to indicate that the collapse of each exocytotic vesicle is slowed by previous nearby collapses. The ultimate fate of synaptic vesicle membrane after collapse appeared to be coalescence with the plasma membrane, as the clusters of particles gradually dispersed into surrounding areas during the first second after a stimulus. The membrane retrieval and recycling that reverse this exocytotic sequence have a slower onset, as has been described in previous reports.

Quick-Freezing to Catch the Membrane Changes that Occur During Exocytosis

1977 ◽  
Vol 35 ◽  
pp. 676-679
Author(s):  
J.E. Heuser
Keyword(s):  
Synaptic Vesicle ◽  
Structural Changes ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Frog Neuromuscular Junction ◽  
The Past ◽  
Synaptic Vesicle Exocytosis ◽  
Quick Freezing ◽  
Vesicle Exocytosis ◽  
Membrane Changes

The technique that we have used to capture synaptic vesicle exocytosis at the frog neuromuscular junction - that of quick-freezing muscles followed by freeze fracture (3) or freeze substitution (6) - works sufficiently well now that it may be useful in other sorts of membrane studies, or studies of fast structural changes with the electron microscope. This note reviews the quickfreezing technique we use, and describes its application to the problem of synaptic vesicle exocytosis and recycling at the synapse.Here, many of the membrane changes of interest occur during the brief delay in synaptic transmission, on a time scale of milliseconds or fractions of milliseconds, and leave only traces thereafter. In the past, we have studied these left-over traces in tissues fixed with the standard chemicals for electron microscopy (1), and have inferred from them that vesicles discharge the quanta of neurotransmitters, as the physiologists would predict.

scholarly journals Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction.

1984 ◽  
Vol 98 (2) ◽  
pp. 685-698 ◽  
Author(s):  
T M Miller ◽  
J E Heuser
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Thin Section ◽  
Nerve Terminal ◽  
Motor Nerve ◽  
Freeze Fracture ◽  
Frog Neuromuscular Junction ◽  
Vesicle Membrane ◽  
Coated Pits ◽  
Active Zones

Frog nerve-muscle preparations were quick-frozen at various times after a single electrical stimulus in the presence of 4-aminopyridine (4-AP), after which motor nerve terminals were visualized by freeze-fracture. Previous studies have shown that such stimulation causes prompt discharge of 3,000-6,000 synaptic vesicles from each nerve terminal and, as a result, adds a large amount of synaptic vesicle membrane to its plasmalemma. In the current experiments, we sought to visualize the endocytic retrieval of this vesicle membrane back into the terminal, during the interval between 1 s and 2 min after stimulation. Two distinct types of endocytosis were observed. The first appeared to be rapid and nonselective. Within the first few seconds after stimulation, relatively large vacuoles (approximately 0.1 micron) pinched off from the plasma membrane, both near to and far away from the active zones. Previous thin-section studies have shown that such vacuoles are not coated with clathrin at any stage during their formation. The second endocytic process was slower and appeared to be selective, because it internalized large intramembrane particles. This process was manifest first by the formation of relatively small (approximately 0.05 micron) indentations in the plasma membrane, which occurred everywhere except at the active zones. These indentations first appeared at 1 s, reached a peak abundance of 5.5/micron2 by 30 s after the stimulus, and disappeared almost completely by 90 s. Previous thin-section studies indicate that these indentations correspond to clathrin-coated pits. Their total abundance is comparable with the number of vesicles that were discharged initially. These endocytic structures could be classified into four intermediate forms, whose relative abundance over time suggests that, at this type of nerve terminal, endocytosis of coated vesicles has the following characteristics: (a) the single endocytotic event is short lived relative to the time scale of two minutes; (b) earlier forms last longer than later forms; and (c) a single event spends a smaller portion of its lifetime in the flat configuration soon after the stimulus than it does later on.

scholarly journals Partial purification of presynaptic plasma membrane by immunoadsorption.

1982 ◽  
Vol 94 (1) ◽  
pp. 88-96 ◽  
Author(s):  
G P Miljanich ◽  
A R Brasier ◽  
R B Kelly
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Nerve Terminal ◽  
Specific Activity ◽  
Electric Organ ◽  
Partial Purification ◽  
Vesicle Membrane ◽  
Specific Activities ◽  
Active Zones ◽  
Presynaptic Plasma Membrane

During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or "active zones." In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased approximately 30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.

scholarly journals Diurnal Changes in Exocytosis and the Number of Synaptic Ribbons at Active Zones of an on-Type Bipolar Cell Terminal

2006 ◽  
Vol 96 (4) ◽  
pp. 2025-2033 ◽  
Author(s):  
Court Hull ◽  
Keith Studholme ◽  
Stephen Yazulla ◽  
Henrique von Gersdorff
Keyword(s):  
Synaptic Vesicle ◽  
Bipolar Cell ◽  
Bipolar Cells ◽  
Synaptic Ribbons ◽  
Diurnal Changes ◽  
Synaptic Vesicle Exocytosis ◽  
Pulse Ratio ◽  
Vesicle Exocytosis ◽  
Active Zones ◽  
Vesicle Pool

The number and morphology of synaptic ribbons at photoreceptor and bipolar cell terminals has been reported to change on a circadian cycle. Here we sought to determine whether this phenomenon exists at goldfish Mb-type bipolar cell terminals with the aim of exploring the role of ribbons in transmitter release. We examined the physiology and ultrastructure of this terminal around two time points: midday and midnight. Nystatin perforated-patch recordings of membrane capacitance ( Cm) revealed that synaptic vesicle exocytosis evoked by short depolarizations was reduced at night, even though Ca2+ currents were larger. The efficiency of exocytosis (measured as the Δ Cm jump per total Ca2+ charge influx) was thus significantly lower at night. The paired-pulse ratio remained unchanged, however, suggesting that release probability was not altered. Hence the decreased exocytosis likely reflects a smaller readily releasable vesicle pool at night. Electron microscopy of single sections from intact retinas averaged 65% fewer ribbons at night. Interestingly, the number of active zones did not change from day to night, only the probability of finding a ribbon at an active zone. Additionally, synaptic vesicle halos surrounding the ribbons were more completely filled at night when these on-type bipolar cells are more hyperpolarized. There was no change, however, in the physical dimensions of synaptic ribbons from day to night. These results suggest that the size of the readily releasable vesicle pool and the efficiency of exocytosis are reduced at night when fewer ribbons are present at bipolar cell terminal active zones.

scholarly journals Botulinum Neurotoxin a Blocks Synaptic Vesicle Exocytosis but Not Endocytosis at the Nerve Terminal

1999 ◽  
Vol 147 (6) ◽  
pp. 1249-1260 ◽  
Author(s):  
Elaine A. Neale ◽  
Linda M. Bowers ◽  
Min Jia ◽  
Karen E. Bateman ◽  
Lura C. Williamson
Keyword(s):  
Synaptic Vesicle ◽  
Nerve Terminal ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Botulinum Neurotoxin ◽  
Vesicle Membrane ◽  
Botulinum Neurotoxin A ◽  
Synaptic Vesicle Exocytosis ◽  
Vesicle Exocytosis ◽  
Botulinum Neurotoxins

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K+-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K+ depolarization, in the presence of Ca2+, triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A–blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca2+ is required for synaptic vesicle membrane retrieval.

scholarly journals Measuring Ca2+-Induced Structural Changes in Lipid Monolayers: Implications for Synaptic Vesicle Exocytosis

2012 ◽  
Vol 102 (6) ◽  
pp. 1394-1402 ◽  
Author(s):  
Sajal Kumar Ghosh ◽  
Simon Castorph ◽  
Oleg Konovalov ◽  
Tim Salditt ◽  
Reinhard Jahn ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Structural Changes ◽  
Lipid Monolayers ◽  
Synaptic Vesicle Exocytosis ◽  
Vesicle Exocytosis

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 Effect of intravenous anesthetic propofol on synaptic vesicle exocytosis at the frog neuromuscular junction

2010 ◽  
Vol 32 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Luciana Ferreira Leite ◽  
Renato Santiago Gomez ◽  
Matheus de Castro Fonseca ◽  
Marcus Vinicius Gomez ◽  
Cristina Guatimosim
Keyword(s):  
Neuromuscular Junction ◽  
Synaptic Vesicle ◽  
Frog Neuromuscular Junction ◽  
Intravenous Anesthetic ◽  
Synaptic Vesicle Exocytosis ◽  
Vesicle Exocytosis

scholarly journals Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.

1979 ◽  
Vol 81 (2) ◽  
pp. 275-300 ◽  
Author(s):  
J E Heuser ◽  
T S Reese ◽  
M J Dennis ◽  
Y Jan ◽  
L Jan ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Neuromuscular Junctions ◽  
Freeze Fracture ◽  
Nerve Impulse ◽  
Biological Tissues ◽  
Synaptic Vesicle Exocytosis ◽  
Capacitance Method ◽  
Quick Freezing ◽  
Vesicle Exocytosis ◽  
Cold Metal

We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the "active zones" that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick-freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the "temporal resolution" of our quick-freezing technique is 2 ms or better.

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