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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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Destaining of weakly labeled boutons is discontinuous during mild depolarization. (A) Ensemble average of boutons labeled as in Fig. 1 D with destaining stimulated by 12 mM K+. Data from 115 boutons from four coverslips. (B) Fluorescence traces from individual boutons reveal stepwise drops in fluorescence intensity. (C) A gallery of large (>14 fluorescence units) stepwise destaining events. (D) A gallery of small (<10 fluorescence units) stepwise destaining events.
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fig2: Destaining of weakly labeled boutons is discontinuous during mild depolarization. (A) Ensemble average of boutons labeled as in Fig. 1 D with destaining stimulated by 12 mM K+. Data from 115 boutons from four coverslips. (B) Fluorescence traces from individual boutons reveal stepwise drops in fluorescence intensity. (C) A gallery of large (>14 fluorescence units) stepwise destaining events. (D) A gallery of small (<10 fluorescence units) stepwise destaining events.

Mentions: To maximize the resolution of destaining events, we monitored bouton destaining during mild (12 mM) depolarization with long acquisition periods (2 s). Fig. 2 A shows that this level of depolarization gives rise to a modest decrease in fluorescence intensity when averaged across boutons. When we examined the destaining profiles of individual boutons a different picture emerged. As illustrated in Fig. 2 B, the fluorescence in individual boutons remained flat for lengthy periods interspersed with abrupt drops in intensity. Although some of these abrupt drops in brightness matched our estimates for the fluorescence intensity of single vesicles (Fig. 2 C), other events were clearly of smaller amplitude (Fig. 2 D). A complete distribution of events (signal + noise) is provided in Fig. S4 B (available at http://www.jcb.org/cgi/content/full/jcb.200407148/DC1). The analysis procedure is described in detail in the online supplemental material.


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)

Destaining of weakly labeled boutons is discontinuous during mild depolarization. (A) Ensemble average of boutons labeled as in Fig. 1 D with destaining stimulated by 12 mM K+. Data from 115 boutons from four coverslips. (B) Fluorescence traces from individual boutons reveal stepwise drops in fluorescence intensity. (C) A gallery of large (>14 fluorescence units) stepwise destaining events. (D) A gallery of small (<10 fluorescence units) stepwise destaining events.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171786&req=5

fig2: Destaining of weakly labeled boutons is discontinuous during mild depolarization. (A) Ensemble average of boutons labeled as in Fig. 1 D with destaining stimulated by 12 mM K+. Data from 115 boutons from four coverslips. (B) Fluorescence traces from individual boutons reveal stepwise drops in fluorescence intensity. (C) A gallery of large (>14 fluorescence units) stepwise destaining events. (D) A gallery of small (<10 fluorescence units) stepwise destaining events.
Mentions: To maximize the resolution of destaining events, we monitored bouton destaining during mild (12 mM) depolarization with long acquisition periods (2 s). Fig. 2 A shows that this level of depolarization gives rise to a modest decrease in fluorescence intensity when averaged across boutons. When we examined the destaining profiles of individual boutons a different picture emerged. As illustrated in Fig. 2 B, the fluorescence in individual boutons remained flat for lengthy periods interspersed with abrupt drops in intensity. Although some of these abrupt drops in brightness matched our estimates for the fluorescence intensity of single vesicles (Fig. 2 C), other events were clearly of smaller amplitude (Fig. 2 D). A complete distribution of events (signal + noise) is provided in Fig. S4 B (available at http://www.jcb.org/cgi/content/full/jcb.200407148/DC1). The analysis procedure is described in detail in the online supplemental material.

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