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Two modes of exocytosis at hippocampal synapses revealed by rate of FM1-43 efflux from individual vesicles.

Richards DA, Bai J, Chapman ER - J. Cell Biol. (2005)

Bottom Line: We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons.These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis.Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin-Madison, Madison, WI 53706, USA.

ABSTRACT
We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons. Two populations of exocytic events were observed; small amplitude events that lose dye slowly, which made up more than half of all events, and faster, larger amplitude events with a fluorescence intensity equivalent to single stained synaptic vesicles. These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis. Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution. Kinetic analysis of the association and dissociation of FM1-43 with membranes, in combination with a simple pore permeation model, indicates that the small, slowly destaining events may be mediated by a narrow approximately 1-nm fusion pore.

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Traces from individual boutons show both large and small events. (A) 62 boutons showing at least two events were analyzed. The amplitude of the first stepwise destaining event was plotted against that of the second. Large and small events were distinguished by a threshold of 14 fluorescence units, indicated by the bold lines; this separated events into four quadrants, numbered 1 through 4, which were then quantified in B. (B) Events were seen in all four quadrants from A. Quadrant 3, small events followed by a second small event, was the most prominent, but occurred in line with the greater proportion of small events seen under these conditions. Overall, the expected distribution would be Q3 > Q1 = Q4 > Q2, and it is not significantly different from this, although the difference between Q3 and the other quadrants is the only one that is significantly different. Error bars are SEM.
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fig4: Traces from individual boutons show both large and small events. (A) 62 boutons showing at least two events were analyzed. The amplitude of the first stepwise destaining event was plotted against that of the second. Large and small events were distinguished by a threshold of 14 fluorescence units, indicated by the bold lines; this separated events into four quadrants, numbered 1 through 4, which were then quantified in B. (B) Events were seen in all four quadrants from A. Quadrant 3, small events followed by a second small event, was the most prominent, but occurred in line with the greater proportion of small events seen under these conditions. Overall, the expected distribution would be Q3 > Q1 = Q4 > Q2, and it is not significantly different from this, although the difference between Q3 and the other quadrants is the only one that is significantly different. Error bars are SEM.

Mentions: Because GABAergic and glutamatergic nerve terminals differ in their exocytic apparatus (Rosenmund et al., 2002), different kinds of boutons might underlie the two populations of release events seen in our experiments. We investigated this possibility by asking whether or not individual boutons showed both small and large exocytic events. We looked at the first two events from boutons, which showed at least two destaining steps, and plotted the amplitude of the first event against that of the second event (Fig. 4 A). All four possibilities were observed, as quantified on a quadrant basis in Fig. 4 B. No clear trends were evident, although individual boutons may have a different overall balance of small and large events. This finding indicates that the two populations of destaining events are not due to heterogeneity of bouton type. It should be noted that, in contrast to the work by Aravanis et al. (2003b), this likely reflects exocytosis of separate vesicles, not repeated exocytosis of a single vesicle.


Two modes of exocytosis at hippocampal synapses revealed by rate of FM1-43 efflux from individual vesicles.

Richards DA, Bai J, Chapman ER - J. Cell Biol. (2005)

Traces from individual boutons show both large and small events. (A) 62 boutons showing at least two events were analyzed. The amplitude of the first stepwise destaining event was plotted against that of the second. Large and small events were distinguished by a threshold of 14 fluorescence units, indicated by the bold lines; this separated events into four quadrants, numbered 1 through 4, which were then quantified in B. (B) Events were seen in all four quadrants from A. Quadrant 3, small events followed by a second small event, was the most prominent, but occurred in line with the greater proportion of small events seen under these conditions. Overall, the expected distribution would be Q3 > Q1 = Q4 > Q2, and it is not significantly different from this, although the difference between Q3 and the other quadrants is the only one that is significantly different. Error bars are SEM.
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Related In: Results  -  Collection

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fig4: Traces from individual boutons show both large and small events. (A) 62 boutons showing at least two events were analyzed. The amplitude of the first stepwise destaining event was plotted against that of the second. Large and small events were distinguished by a threshold of 14 fluorescence units, indicated by the bold lines; this separated events into four quadrants, numbered 1 through 4, which were then quantified in B. (B) Events were seen in all four quadrants from A. Quadrant 3, small events followed by a second small event, was the most prominent, but occurred in line with the greater proportion of small events seen under these conditions. Overall, the expected distribution would be Q3 > Q1 = Q4 > Q2, and it is not significantly different from this, although the difference between Q3 and the other quadrants is the only one that is significantly different. Error bars are SEM.
Mentions: Because GABAergic and glutamatergic nerve terminals differ in their exocytic apparatus (Rosenmund et al., 2002), different kinds of boutons might underlie the two populations of release events seen in our experiments. We investigated this possibility by asking whether or not individual boutons showed both small and large exocytic events. We looked at the first two events from boutons, which showed at least two destaining steps, and plotted the amplitude of the first event against that of the second event (Fig. 4 A). All four possibilities were observed, as quantified on a quadrant basis in Fig. 4 B. No clear trends were evident, although individual boutons may have a different overall balance of small and large events. This finding indicates that the two populations of destaining events are not due to heterogeneity of bouton type. It should be noted that, in contrast to the work by Aravanis et al. (2003b), this likely reflects exocytosis of separate vesicles, not repeated exocytosis of a single vesicle.

Bottom Line: We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons.These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis.Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin-Madison, Madison, WI 53706, USA.

ABSTRACT
We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons. Two populations of exocytic events were observed; small amplitude events that lose dye slowly, which made up more than half of all events, and faster, larger amplitude events with a fluorescence intensity equivalent to single stained synaptic vesicles. These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis. Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution. Kinetic analysis of the association and dissociation of FM1-43 with membranes, in combination with a simple pore permeation model, indicates that the small, slowly destaining events may be mediated by a narrow approximately 1-nm fusion pore.

Show MeSH
Related in: MedlinePlus