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.

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 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 Role of specific presynaptic calcium channel subtypes in isoflurane inhibition of synaptic vesicle exocytosis in rat hippocampal neurones

2019 ◽  
Vol 123 (2) ◽  
pp. 219-227 ◽  
Author(s):  
Yuko Koyanagi ◽  
Christina L. Torturo ◽  
Daniel C. Cook ◽  
Zhenyu Zhou ◽  
Hugh C. Hemmings
Keyword(s):  
Calcium Channel ◽  
Synaptic Vesicle ◽  
Synaptic Vesicle Exocytosis ◽  
Vesicle Exocytosis ◽  
Presynaptic Calcium

scholarly journals SNARE Complex Oligomerization by Synaphin/Complexin Is Essential for Synaptic Vesicle Exocytosis

Cell ◽  
2001 ◽  
Vol 104 (3) ◽  
pp. 421-432 ◽  
Author(s):  
Hiroshi Tokumaru ◽  
Keiko Umayahara ◽  
Lorenzo L Pellegrini ◽  
Toru Ishizuka ◽  
Hideo Saisu ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Snare Complex ◽  
Synaptic Vesicle Exocytosis ◽  
Vesicle Exocytosis

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.

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