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Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles.

Leitz J, Kavalali ET - Elife (2014)

Bottom Line: Presynaptic terminals release neurotransmitters spontaneously in a manner that can be regulated by Ca(2+).However, the mechanisms underlying this regulation are poorly understood because the inherent stochasticity and low probability of spontaneous fusion events has curtailed their visualization at individual release sites.Here, using pH-sensitive optical probes targeted to synaptic vesicles, we visualized single spontaneous fusion events and found that they are retrieved extremely rapidly with faster re-acidification kinetics than their action potential-evoked counterparts.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, United States.

ABSTRACT
Presynaptic terminals release neurotransmitters spontaneously in a manner that can be regulated by Ca(2+). However, the mechanisms underlying this regulation are poorly understood because the inherent stochasticity and low probability of spontaneous fusion events has curtailed their visualization at individual release sites. Here, using pH-sensitive optical probes targeted to synaptic vesicles, we visualized single spontaneous fusion events and found that they are retrieved extremely rapidly with faster re-acidification kinetics than their action potential-evoked counterparts. These fusion events were coupled to postsynaptic NMDA receptor-driven Ca(2+) signals, and at elevated Ca(2+) concentrations there was an increase in the number of vesicles that would undergo fusion. Furthermore, spontaneous vesicle fusion propensity in a synapse was Ca(2+)-dependent but regulated autonomously: independent of evoked fusion probability at the same synapse. Taken together, these results expand classical quantal analysis to incorporate endocytic and exocytic phases of single fusion events and uncover autonomous regulation of spontaneous fusion.

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Increasing extracellular Ca2+ does not alter decay time of spontaneous increases in sypHTomato fluorescence.(A) Example average traces of spontaneous increases in sypHTomato fluorescence (red) single experiments in 2 mM (left), 4 mM (middle), and 8 mM (right) extracellular Ca2+ with decay time fit in blue. (B) Example average traces from the same experiments as A of PSD-95-GCaMP5K events (green) that correspond with sypHTomato spontaneous increases in fluorescence in 2 mM (left), 4 mM (middle), and 8 mM (right) extracellular Ca2+ with decay time fit in blue. (C) Decay time does not change as a function of extracellular Ca2+, however increases in fluorescence due to stimulation are slower to decay than spontaneous increases in fluorescence (p < 0.05 One-way ANOVA with Bonferroni post hoc analysis).DOI:http://dx.doi.org/10.7554/eLife.03658.008
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fig4s1: Increasing extracellular Ca2+ does not alter decay time of spontaneous increases in sypHTomato fluorescence.(A) Example average traces of spontaneous increases in sypHTomato fluorescence (red) single experiments in 2 mM (left), 4 mM (middle), and 8 mM (right) extracellular Ca2+ with decay time fit in blue. (B) Example average traces from the same experiments as A of PSD-95-GCaMP5K events (green) that correspond with sypHTomato spontaneous increases in fluorescence in 2 mM (left), 4 mM (middle), and 8 mM (right) extracellular Ca2+ with decay time fit in blue. (C) Decay time does not change as a function of extracellular Ca2+, however increases in fluorescence due to stimulation are slower to decay than spontaneous increases in fluorescence (p < 0.05 One-way ANOVA with Bonferroni post hoc analysis).DOI:http://dx.doi.org/10.7554/eLife.03658.008

Mentions: Our earlier work showed that an increase in the number of vesicles that fuse slows the fluorescence decay time of fusion events (Leitz and Kavalali, 2011). Therefore, here, we analyzed the decay times of events in 8 mM Ca2+ as a function of amplitude and found that there was no correlation in event size and fluorescence decay times (Figure 4D). Furthermore, we also found a similar trend of Ca2+-independence in neurons expressing SypHTomato/PSD-95-GCaMP5K (Figure 4—figure supplement 1A and B). All of these decay times, regardless of extracellular Ca2+ levels, were faster than those observed during stimulation-evoked fusion (Figure 4—figure supplement 1C). It is important to note that the rate of decay is approaching the limit of our temporal resolution, which is lowered in an attempt to reduce photobleaching during long imaging episodes required to identify spontaneous fusion events. Thus, it is possible that we either cannot detect a significant change in decay times, or we are missing a subset of ultra fast decay times. Regardless, these data not only suggest that the mechanisms controlling the rate of synaptic vesicle reacidification are not the same for spontaneous and stimulation-evoked vesicle fusion but that endocytosis of synaptic vesicles released at rest is rapid even during multivesicular fusion events, unlike retrieval of vesicles released in response to stimulation.


Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles.

Leitz J, Kavalali ET - Elife (2014)

Increasing extracellular Ca2+ does not alter decay time of spontaneous increases in sypHTomato fluorescence.(A) Example average traces of spontaneous increases in sypHTomato fluorescence (red) single experiments in 2 mM (left), 4 mM (middle), and 8 mM (right) extracellular Ca2+ with decay time fit in blue. (B) Example average traces from the same experiments as A of PSD-95-GCaMP5K events (green) that correspond with sypHTomato spontaneous increases in fluorescence in 2 mM (left), 4 mM (middle), and 8 mM (right) extracellular Ca2+ with decay time fit in blue. (C) Decay time does not change as a function of extracellular Ca2+, however increases in fluorescence due to stimulation are slower to decay than spontaneous increases in fluorescence (p < 0.05 One-way ANOVA with Bonferroni post hoc analysis).DOI:http://dx.doi.org/10.7554/eLife.03658.008
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4270043&req=5

fig4s1: Increasing extracellular Ca2+ does not alter decay time of spontaneous increases in sypHTomato fluorescence.(A) Example average traces of spontaneous increases in sypHTomato fluorescence (red) single experiments in 2 mM (left), 4 mM (middle), and 8 mM (right) extracellular Ca2+ with decay time fit in blue. (B) Example average traces from the same experiments as A of PSD-95-GCaMP5K events (green) that correspond with sypHTomato spontaneous increases in fluorescence in 2 mM (left), 4 mM (middle), and 8 mM (right) extracellular Ca2+ with decay time fit in blue. (C) Decay time does not change as a function of extracellular Ca2+, however increases in fluorescence due to stimulation are slower to decay than spontaneous increases in fluorescence (p < 0.05 One-way ANOVA with Bonferroni post hoc analysis).DOI:http://dx.doi.org/10.7554/eLife.03658.008
Mentions: Our earlier work showed that an increase in the number of vesicles that fuse slows the fluorescence decay time of fusion events (Leitz and Kavalali, 2011). Therefore, here, we analyzed the decay times of events in 8 mM Ca2+ as a function of amplitude and found that there was no correlation in event size and fluorescence decay times (Figure 4D). Furthermore, we also found a similar trend of Ca2+-independence in neurons expressing SypHTomato/PSD-95-GCaMP5K (Figure 4—figure supplement 1A and B). All of these decay times, regardless of extracellular Ca2+ levels, were faster than those observed during stimulation-evoked fusion (Figure 4—figure supplement 1C). It is important to note that the rate of decay is approaching the limit of our temporal resolution, which is lowered in an attempt to reduce photobleaching during long imaging episodes required to identify spontaneous fusion events. Thus, it is possible that we either cannot detect a significant change in decay times, or we are missing a subset of ultra fast decay times. Regardless, these data not only suggest that the mechanisms controlling the rate of synaptic vesicle reacidification are not the same for spontaneous and stimulation-evoked vesicle fusion but that endocytosis of synaptic vesicles released at rest is rapid even during multivesicular fusion events, unlike retrieval of vesicles released in response to stimulation.

Bottom Line: Presynaptic terminals release neurotransmitters spontaneously in a manner that can be regulated by Ca(2+).However, the mechanisms underlying this regulation are poorly understood because the inherent stochasticity and low probability of spontaneous fusion events has curtailed their visualization at individual release sites.Here, using pH-sensitive optical probes targeted to synaptic vesicles, we visualized single spontaneous fusion events and found that they are retrieved extremely rapidly with faster re-acidification kinetics than their action potential-evoked counterparts.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, United States.

ABSTRACT
Presynaptic terminals release neurotransmitters spontaneously in a manner that can be regulated by Ca(2+). However, the mechanisms underlying this regulation are poorly understood because the inherent stochasticity and low probability of spontaneous fusion events has curtailed their visualization at individual release sites. Here, using pH-sensitive optical probes targeted to synaptic vesicles, we visualized single spontaneous fusion events and found that they are retrieved extremely rapidly with faster re-acidification kinetics than their action potential-evoked counterparts. These fusion events were coupled to postsynaptic NMDA receptor-driven Ca(2+) signals, and at elevated Ca(2+) concentrations there was an increase in the number of vesicles that would undergo fusion. Furthermore, spontaneous vesicle fusion propensity in a synapse was Ca(2+)-dependent but regulated autonomously: independent of evoked fusion probability at the same synapse. Taken together, these results expand classical quantal analysis to incorporate endocytic and exocytic phases of single fusion events and uncover autonomous regulation of spontaneous fusion.

Show MeSH
Related in: MedlinePlus