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 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.

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.

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 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

Use of Aldehyde Fixatives to Determine the Rate of Synaptic Transmitter Release

1980 ◽  
Vol 89 (1) ◽  
pp. 19-28
Author(s):  
J. E. SMITH ◽  
T. S. REESE
Keyword(s):  
Synaptic Vesicle ◽  
Transmitter Release ◽  
Morphological Changes ◽  
Freeze Fracture ◽  
Magnesium Concentration ◽  
Synaptic Vesicle Exocytosis ◽  
Aldehyde Fixation ◽  
Vesicle Exocytosis ◽  
Nerve Muscle ◽  
External Calcium

Aldehyde fixation continues to be useful to prepare synapses for freeze-fracture, but it may increase the rate of transmitter release. The effects of different aldehyde fixatives on spontaneous quantal release (m.e.p.p.s), and on the corresponding synaptic vesicle exocytosis at frog nerve-muscle synapses were investigated with the hope of finding a way to minimize side effects of fixation. Increases in m.e.p.p.s of up to 50 s−1 occurred during fixation, despite the species of aldehyde used in the fixative, and this fixative effect decreased only slightly as aldehyde concentration was increased. Increases in m.e.p.p. frequency were not blocked by tetrodotoxin, by lowering external calcium and raising external magnesium concentration, or by lowering the total osmotic strength of the fixative. The smallest increase in m.e.p.p. frequency was in 3% glutaraldehyde and corresponded to the lowest level of synaptic vesicle exocytosis seen by freeze-fracture, 0·15per μm of active zone. The effects of aldehyde fixation on m.e.p.p. frequency and synaptic vesicle exocytosis could not be avoided, but this study suggests how its effect on morphological changes in synapses might be minimized.

How to sample the entire length of a single muscle fiber quick-frozen after electrical point stimulation for high resolution EM

1992 ◽  
Vol 50 (1) ◽  
pp. 776-777
Author(s):  
Joachim R. Sommer ◽  
Teresa High ◽  
Betty Scherer ◽  
Isaiah Taylor ◽  
Rashid Nassar
Keyword(s):  
Skeletal Muscle ◽  
High Resolution ◽  
Action Potential ◽  
Muscle Fiber ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Electrical Field Stimulation ◽  
Skeletal Muscle Fiber ◽  
Freeze Dried ◽  
Quick Freezing

We have developed a model that allows the quick-freezing at known time intervals following electrical field stimulation of a single, intact frog skeletal muscle fiber isolated by sharp dissection. The preparation is used for studying high resolution morphology by freeze-substitution and freeze-fracture and for electron probe x-ray microanlysis of sudden calcium displacement from intracellular stores in freeze-dried cryosections, all in the same fiber. We now show the feasibility and instrumentation of new methodology for stimulating a single, intact skeletal muscle fiber at a point resulting in the propagation of an action potential, followed by quick-freezing with sub-millisecond temporal resolution after electrical stimulation, followed by multiple sampling of the frozen muscle fiber for freeze-substitution, freeze-fracture (not shown) and cryosectionmg. This model, at once serving as its own control and obviating consideration of variances between different fibers, frogs etc., is useful to investigate structural and topochemical alterations occurring in the wake of an action potential.

The consolidation of microscopy technology into a university research support facility: Reflections on the 1980's and projections for the 1990's at the university of iowa

1993 ◽  
Vol 51 ◽  
pp. 476-477
Author(s):  
Kenneth C. Moore
Keyword(s):  
Laser Scanning ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Research Support ◽  
University Of Iowa ◽  
Staff Members ◽  
The Past ◽  
Research Grade ◽  
The University ◽  
Support Facility

The University of Iowa Central Electron Microscopy Research Facility(CEMRF) was established in 1981 to support all faculty, staff and students needing this technology. Initially the CEMRF was operated with one TEM, one SEM, three staff members and supported about 30 projects a year. During the past twelve years, the facility has replaced all instrumentation pre-dating 1981, and now includes 2 TEM's, 2 SEM's, 2 EDS systems, cryo-transfer specimen holders for both TEM and SEM, 2 parafin microtomes, 4 ultamicrotomes including cryoultramicrotomy, a Laser Scanning Confocal microscope, a research grade light microscope, an Ion Mill, film and print processing equipment, a rapid cryo-freezer, freeze substitution apparatus, a freeze-fracture/etching system, vacuum evaporators, sputter coaters, a plasma asher, and is currently evaluating scanning probe microscopes for acquisition. The facility presently consists of 10 staff members and supports over 150 projects annually from 44 departments in 5 Colleges and 10 industrial laboratories. One of the unique strengths of the CEMRF is that both Biomedical and Physical scientists use the facility.

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