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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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Small and large events lose their dye contents with different kinetics. (A) Example traces of boutons labeled with 25 mM K+ for 1 min, and then subsequently stimulated with 25 mM K+. Imaging frequency was 2.5 frames per second. The abrupt drops in fluorescence intensity no longer occur in a strictly stepwise fashion. 162 events from ∼300 boutons were analyzed in B–E. (B) The exocytic events were fit with single exponentials. τ values are plotted against frequency, and once more exhibit two apparent populations. (C) The amplitude of the drops in fluorescence are plotted against frequency as in Fig. 3 (A and B), and provide a similar pattern. (D) Large (>14 units; open circles) and small amplitude (<14 units; closed circles) events were time aligned and averaged, providing group mean time constants of 0.65 ± 0.04 and 3.11 ± 0.12 s, respectively. (E) The rate constant (1/τ) is plotted against the amplitude of the fluorescence drop. Small events are tightly clustered in both rate constant and amplitude, whereas large events are more variable especially in rate constant. (F) If considered more rigorously, small amplitude events would be expected, according to our hypothesis, to show a behavior where they initially present an exponential decline, which is truncated before completion (by fusion pore closure, in our model). To demonstrate the effect of this on our simple exponential fits (D and E), we have plotted a small amplitude event with an average time course (2.6 s). When we make the assumption that had destaining continued to completion it would have reached a net fluorescence drop of 16 units, and conducted the fit assuming it was interrupted (i.e., by progressive truncation of the trace), we find that the new value for τ is 6.4 s. When performed for all events in this category, we find the mean τ of dye efflux to be 7.16 ± 0.97 s. Error bars are SEM.
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fig5: Small and large events lose their dye contents with different kinetics. (A) Example traces of boutons labeled with 25 mM K+ for 1 min, and then subsequently stimulated with 25 mM K+. Imaging frequency was 2.5 frames per second. The abrupt drops in fluorescence intensity no longer occur in a strictly stepwise fashion. 162 events from ∼300 boutons were analyzed in B–E. (B) The exocytic events were fit with single exponentials. τ values are plotted against frequency, and once more exhibit two apparent populations. (C) The amplitude of the drops in fluorescence are plotted against frequency as in Fig. 3 (A and B), and provide a similar pattern. (D) Large (>14 units; open circles) and small amplitude (<14 units; closed circles) events were time aligned and averaged, providing group mean time constants of 0.65 ± 0.04 and 3.11 ± 0.12 s, respectively. (E) The rate constant (1/τ) is plotted against the amplitude of the fluorescence drop. Small events are tightly clustered in both rate constant and amplitude, whereas large events are more variable especially in rate constant. (F) If considered more rigorously, small amplitude events would be expected, according to our hypothesis, to show a behavior where they initially present an exponential decline, which is truncated before completion (by fusion pore closure, in our model). To demonstrate the effect of this on our simple exponential fits (D and E), we have plotted a small amplitude event with an average time course (2.6 s). When we make the assumption that had destaining continued to completion it would have reached a net fluorescence drop of 16 units, and conducted the fit assuming it was interrupted (i.e., by progressive truncation of the trace), we find that the new value for τ is 6.4 s. When performed for all events in this category, we find the mean τ of dye efflux to be 7.16 ± 0.97 s. Error bars are SEM.

Mentions: If vesicles undergo full collapse into the plasma membrane they would be expected to lose their dye at a rate limited only by the lateral diffusion within the membrane and the departitioning kinetics of the dye, whereas permeation through a narrow fusion pore might be expected to significantly slow the rate of efflux of the dye. To follow the dye efflux rate, we increased the pixel binning and imaged at 400 ms per time point. We also increased the depolarizing stimulus to 25 mM K+, which provided a sufficient number of destaining events while still not causing temporally overlapping events at individual boutons. As we show in Fig. 5 A, although some boutons did not release dye during the stimulus period, others showed clear and abrupt diminutions in brightness. When examined closely, these could be seen to no longer have the step function seen in previous experiments; rather, they showed a more gradual reduction in intensity. These individual destaining events were fitted to single exponentials. The distribution of τ values obtained from such fits are plotted in Fig. 5 B, and the distribution of amplitudes is plotted in Fig. 5 C. In the case of both τ and amplitude, the results fell into two groups, suggesting that two exocytic modes underlie release at these synapses. We were able to separate them with a threshold of 14 fluorescence units, and measure the time course of averaged events (Fig. 5 D), providing average values of τ = 0.65 ± 0.04 s for large amplitude events and τ = 3.11 ± 0.12 s for small events, indicating that they are kinetically distinct.


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)

Small and large events lose their dye contents with different kinetics. (A) Example traces of boutons labeled with 25 mM K+ for 1 min, and then subsequently stimulated with 25 mM K+. Imaging frequency was 2.5 frames per second. The abrupt drops in fluorescence intensity no longer occur in a strictly stepwise fashion. 162 events from ∼300 boutons were analyzed in B–E. (B) The exocytic events were fit with single exponentials. τ values are plotted against frequency, and once more exhibit two apparent populations. (C) The amplitude of the drops in fluorescence are plotted against frequency as in Fig. 3 (A and B), and provide a similar pattern. (D) Large (>14 units; open circles) and small amplitude (<14 units; closed circles) events were time aligned and averaged, providing group mean time constants of 0.65 ± 0.04 and 3.11 ± 0.12 s, respectively. (E) The rate constant (1/τ) is plotted against the amplitude of the fluorescence drop. Small events are tightly clustered in both rate constant and amplitude, whereas large events are more variable especially in rate constant. (F) If considered more rigorously, small amplitude events would be expected, according to our hypothesis, to show a behavior where they initially present an exponential decline, which is truncated before completion (by fusion pore closure, in our model). To demonstrate the effect of this on our simple exponential fits (D and E), we have plotted a small amplitude event with an average time course (2.6 s). When we make the assumption that had destaining continued to completion it would have reached a net fluorescence drop of 16 units, and conducted the fit assuming it was interrupted (i.e., by progressive truncation of the trace), we find that the new value for τ is 6.4 s. When performed for all events in this category, we find the mean τ of dye efflux to be 7.16 ± 0.97 s. Error bars are SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171786&req=5

fig5: Small and large events lose their dye contents with different kinetics. (A) Example traces of boutons labeled with 25 mM K+ for 1 min, and then subsequently stimulated with 25 mM K+. Imaging frequency was 2.5 frames per second. The abrupt drops in fluorescence intensity no longer occur in a strictly stepwise fashion. 162 events from ∼300 boutons were analyzed in B–E. (B) The exocytic events were fit with single exponentials. τ values are plotted against frequency, and once more exhibit two apparent populations. (C) The amplitude of the drops in fluorescence are plotted against frequency as in Fig. 3 (A and B), and provide a similar pattern. (D) Large (>14 units; open circles) and small amplitude (<14 units; closed circles) events were time aligned and averaged, providing group mean time constants of 0.65 ± 0.04 and 3.11 ± 0.12 s, respectively. (E) The rate constant (1/τ) is plotted against the amplitude of the fluorescence drop. Small events are tightly clustered in both rate constant and amplitude, whereas large events are more variable especially in rate constant. (F) If considered more rigorously, small amplitude events would be expected, according to our hypothesis, to show a behavior where they initially present an exponential decline, which is truncated before completion (by fusion pore closure, in our model). To demonstrate the effect of this on our simple exponential fits (D and E), we have plotted a small amplitude event with an average time course (2.6 s). When we make the assumption that had destaining continued to completion it would have reached a net fluorescence drop of 16 units, and conducted the fit assuming it was interrupted (i.e., by progressive truncation of the trace), we find that the new value for τ is 6.4 s. When performed for all events in this category, we find the mean τ of dye efflux to be 7.16 ± 0.97 s. Error bars are SEM.
Mentions: If vesicles undergo full collapse into the plasma membrane they would be expected to lose their dye at a rate limited only by the lateral diffusion within the membrane and the departitioning kinetics of the dye, whereas permeation through a narrow fusion pore might be expected to significantly slow the rate of efflux of the dye. To follow the dye efflux rate, we increased the pixel binning and imaged at 400 ms per time point. We also increased the depolarizing stimulus to 25 mM K+, which provided a sufficient number of destaining events while still not causing temporally overlapping events at individual boutons. As we show in Fig. 5 A, although some boutons did not release dye during the stimulus period, others showed clear and abrupt diminutions in brightness. When examined closely, these could be seen to no longer have the step function seen in previous experiments; rather, they showed a more gradual reduction in intensity. These individual destaining events were fitted to single exponentials. The distribution of τ values obtained from such fits are plotted in Fig. 5 B, and the distribution of amplitudes is plotted in Fig. 5 C. In the case of both τ and amplitude, the results fell into two groups, suggesting that two exocytic modes underlie release at these synapses. We were able to separate them with a threshold of 14 fluorescence units, and measure the time course of averaged events (Fig. 5 D), providing average values of τ = 0.65 ± 0.04 s for large amplitude events and τ = 3.11 ± 0.12 s for small events, indicating that they are kinetically distinct.

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