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

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100080808 ◽

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.

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Structural changes after transmitter release at the frog neuromuscular junction.

The Journal of Cell Biology ◽

10.1083/jcb.88.3.564 ◽

1981 ◽

Vol 88 (3) ◽

pp. 564-580 ◽

Cited By ~ 309

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.

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

Journal of Experimental Biology ◽

10.1242/jeb.89.1.19 ◽

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.

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Intracellular Acidification Suppresses Synaptic Vesicle Mobilization in the Motor Nerve Terminals

Acta Naturae ◽

10.32607/actanaturae.11054 ◽

2020 ◽

Vol 12 (4) ◽

pp. 105-113

Author(s):

A. L. Zefirov ◽

R. D. Mukhametzyanov ◽

A. V. Zakharov ◽

K. A. Mukhutdinova ◽

U. O. Odnoshivkina ◽

...

Keyword(s):

Synaptic Vesicle ◽

Neurotransmitter Release ◽

Neuromuscular Junctions ◽

Motor Nerve ◽

Synaptic Activity ◽

Special Role ◽

Intracellular Acidification ◽

Synaptic Vesicle Exocytosis ◽

Vesicle Exocytosis ◽

Cytoplasmic Acidification

Intracellular protons play a special role in the regulation of presynaptic processes, since the functioning of synaptic vesicles and endosomes depends on their acidification by the H+-pump. Furthermore, transient acidification of the intraterminal space occurs during synaptic activity. Using microelectrode recording of postsynaptic responses (an indicator of neurotransmitter release) and exo-endocytic marker FM1-43, we studied the effects of intracellular acidification with propionate on the presynaptic events underlying neurotransmitter release. Cytoplasmic acidification led to a marked decrease in neurotransmitter release during the first minute of a 20-Hz stimulation in the neuromuscular junctions of mouse diaphragm and frog cutaneous pectoris muscle. This was accompanied by a reduction in the FM1-43 loss during synaptic vesicle exocytosis in response to the stimulation. Estimation of the endocytic uptake of FM1-43 showed no disruption in synaptic vesicle endocytosis. Acidification completely prevented the action of the cell-membrane permeable compound 24-hydroxycholesterol, which can enhance synaptic vesicle mobilization. Thus, the obtained results suggest that an increase in [H+]in negatively regulates neurotransmission due to the suppression of synaptic vesicle delivery to the sites of exocytosis at high activity. This mechanism can be a part of the negative feedback loop in regulating neurotransmitter release.

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