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. 2009 Jun;12(6):751-8.
doi: 10.1038/nn.2317. Epub 2009 May 10.

A resting pool of vesicles is responsible for spontaneous vesicle fusion at the synapse

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A resting pool of vesicles is responsible for spontaneous vesicle fusion at the synapse

Naila Ben Fredj et al. Nat Neurosci. 2009 Jun.

Abstract

Synapses relay information through the release of neurotransmitters stored in presynaptic vesicles. The identity, kinetics and location of the vesicle pools that are mobilized by neuronal activity have been studied using a variety of techniques. We created a genetically encoded probe, biosyn, which consists of a biotinylated VAMP2 expressed at presynaptic terminals. We exploited the high-affinity interaction between streptavidin and biotin to label biosyn with fluorescent streptavidin during vesicle fusion. This approach allowed us to tag vesicles sequentially to visualize and establish the identity of presynaptic pools. Using this technique, we were able to distinguish between two different pools of vesicles in rat hippocampal neurons: one that was released in response to presynaptic activity and another, distinct vesicle pool that spontaneously fused with the plasma membrane. We found that the spontaneous vesicles belonged to a 'resting pool' that is normally not mobilized by neuronal activity and whose function was previously unknown.

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Figures

Figure 1
Figure 1. Visualizing vesicles released by membrane depolarization using biosyn
(a) Diagram showing labelling of biosyn with streptavidin. Neurons are first pre-treated with streptavidin Alexa647 (strep647, shown in blue) to quench surface VAMP2. Excess streptavidin is removed by washing. The neurons are subsequently stimulated in the presence of streptavidin Alexa555 (strep555, shown in red) to label the vesicles that fuse with the plasma membrane during neurotransmitter release and expose their intraluminal biosyn molecules to the extracellular space. (b) Schematic diagram showing the timeline of the experimental protocol. Surface biosyn was stained with strep647 (blue bar) for 30 sec., washed and then neurons were stimulated twice with a high K+ solution in the presence of strep555 (red bar; 5 min interval between depolarizations). After washing, neurons were fixed and imaged. (c) Hippocampal neurons were co-transfected with biosyn and synaptophysin-pHluorin (SypHy). Images show co-localisation of individual synapses expressing SypHy (green), with the recycling pool of vesicle labelled with strep555 (red) and surface labelling with strep647 (blue). Scale bar = 10 μm. (d) Graph showing a plot of the fluorescence intensity of synapses labelled by depolarization as a function of surface label. Individual synapses are shown in gray and the binned (groups of 10) values in black (n = 445 synapses from 20 cells). (e) Quantification of biosyn staining under different conditions. High K+ stimulation specifically labels the activity dependent pool when calcium is present. All conditions were normalized to this value. Staining is abolished when calcium is absent or when cadmium (a calcium channel blocker) is present (n > 200 synapses from 10 cells per condition). Values are shown as mean ± SEM.
Figure 2
Figure 2. Biosyn used as a reliable tool to assess evoked vesicle fusion
Hippocampal neurons were co-transfected with biosyn and sypHy. (a) Schematic diagram showing the timeline of the experimental protocol: neurons were stimulated with 900 APs at 20 Hz in the presence of strep555 to label biosyn, while measuring the amount of exocytosis reported by synaptophysin-pHluorin (sypHy) in the green channel. (b) Images show sypHy fluorescence before and immediately after the stimulus. (c) Responses to 900 APs at 20 Hz (black bar) measured from all synapses analyzed from a single cell (gray) with the average response (black) overlayed. (d) After the stimulus and having washed away the strep555, synapses were found to be strongly labelled. Note the red puncta clearly co-localise with sypHy. Scale bar = 5 μm. (e) The graph plots the fluorescence intensity of biosyn for individual synapses as a function of the change in fluorescence measured with sypHy (n = 145 synapses from 6 cells). The black line represents the best linear fit to the data set, constrained to go through the origin. Values are shown as mean ± SEM.
Figure 3
Figure 3. Visualising vesicles released spontaneously with biosyn
(a) Schematic diagram showing the timeline of the experimental protocol. After labelling surface biosyn with strep647 (blue bar) for 30 sec., spontaneous vesicle fusion was labelled with strep555 (red bar) in the presence of TTX, nominally zero calcium and incubated at 37° C for 15 min. After washing, neurons were fixed and imaged. (b) Hippocampal neurons were co-transfected with biosyn and sypHy. Images show individual synapses expressing sypHy (green), spontaneous vesicle fusion events labelled with strep555 (red) and surface biosyn labelled with strep647 (blue). Scale bar = 5 μm. (c) Plot of fluorescence intensity for spontaneous versus surface biosyn for individual synapses (n = 545 synapses from 28 cells). (d) Time course of spontaneous vesicle fusion. Hippocampal neurons were treated with strep555 for different periods of time. The graph shows the fluorescence intensity of spontaneous labelling as a function of time exposed to strep555. The data was fit with the following exponential function: F = FmaxF1 exp(−t/τ), where F is the fluorescence of spontaneous release, Fmax is the maximum fluorescence, t is time and τ is the time constant. We find a pool of vesicles is released spontaneously with a time-constant (τ) of 6.2 min at 37° C (filled circles; n = 18-25 cells per condition). The open circle is a single time point at 15 min performed at room temperature. Values are shown as mean ± SEM.
Figure 4
Figure 4. Two distinct pools of vesicle with different release modes: spontaneous and evoked
(a) Two consecutive depolarisations with strep488 (green) label the entire recycling pool. A further depolarisation with strep594 (red) results in no further labelling. (b) Labelling the recycling pool (green) followed by spontaneous labelling (red) results in a strong signal in both channels, suggesting that each set of vesicles draws from two distinct pools. Scale bar = 5 μm. (c) Example from one set of sister cultures showing the cumulative distribution of fluorescence intensity for synapses labelled with strep594 for spontaneous fusion before (grey line, n = 140 synapses) and after (solid line, n = 185 synapses) depolarization (the median intensity is not significant between the two conditions; one-way ANOVA, p>0.05). The dashed line shows spontaneous labelling after the entire pool of spontaneous vesicles was labelled with another colour (n = 85 synapses; statistically significant when compared to either of the two conditions plotted in the graph; one-way ANOVA, p<0.001). Note that the depletion of the recycling (activity-dependent) pool does not affect the size of the spontaneous pool. (d) Cumulative distribution of fluorescence intensity for synapses from the same sister cultures labelled with strep488 using a depolarizing stimulus before (solid line, n = 185 synapses) and after (grey line, n = 140 synapses) the spontaneous pool is released (the median intensity is not significant between the two conditions; one-way ANOVA, p>0.05). The dashed line shows evoked labelling after the entire pool of evoked vesicles are labelled with another colour (n = 263 synapses; statistically significant when compared to either of the two conditions plotted in the graph; one-way ANOVA, p<0.001). Note that (c) and (d) use different fluorescent probes (strep594 and strep488, respectively). Fluorescence intensities can only be compared between curves of the same graph. Values are shown as mean ± SEM.
Figure 5
Figure 5. Quantification of fluorescence intensity for different conditions
(a) The graph shows the fluorescence intensity of synapses labelled with a depolarizing stimulus (D) or during spontaneous release (S). Each condition involves several labelling protocols, the sequence of which is shown below each bar. The fluorescence intensity plotted corresponds to that shown as the filled squares/rectangles (black), whereas the empty squares/rectangles are treatments that are not plotted above. The first two bars (from the left) show the intensity of vesicles labelled with depolarizing stimuli, delivered before or after another depolarizing stimulus (n = 64 neurons for each bar). The next two bars also represent the intensity of vesicles labelled with depolarizing stimuli, this time before or after spontaneous release (n = 28 and n = 32 neurons, respectively). The next set of four bars corresponds to the intensity of vesicles labelled spontaneously. Regardless of when spontaneous release is assessed (on its own, before or after a depolarizing stimulus) the total intensity of spontaneous labelling is very similar (n = 21, n = 27 and n = 32 neurons, respectively). Note that spontaneous labelling after the spontaneous pool was mobilized is significantly reduced, indicating pool saturation. The final bar is a control to show that surface labelling of biosyn (30 sec exposure to streptavidin) saturates all biosyn binding sites (n = 14 neurons). Statistical analysis was performed using a non-parametric one way a ANOVA test. ns: not significant (p>0.05). (b) Graph showing the fluorescence intensity of the spontaneous pool as a function of the fluorescence intensity of the recycling pool, for individual synapses (gray dots) and grouped in bins of 30 (black circles; n = 989 synapses from 26 cells). Note the significant degree of correlation (correlation coefficient = 0.57, p < 0.05). Values are shown as mean ± SEM.
Figure 6
Figure 6. Measuring vesicle pools with sypHy
(a) Changes in fluorescence from a single neuron in response to 900 APs at 20 Hz to mobilize the entire recycling pool. Unquenching the remaining vesicles with Nh4Cl uncovers the resting pool. (b) The same experiment as in (a) was performed, this time allowing for spontaneous release to occur after the recycling pool has been mobilized. A single neuron was stimulated with 900 APs at 20 Hz to mobilize the entire recycling pool. Synapses were left for 15 min at room temperature in the presence of TTX to establish whether any vesicles could be released spontaneously. Finally, any remaining vesicles were unquenched by addition of Nh4Cl. The size of the recycling pool and the resting pool of vesicles are shown for each cell. (c) Example images of the presynaptic terminals from the neuron shown in (b) after different treatments. Top from left to right: synapses at rest and after 900 APs at 20 Hz; bottom from left to right: synapses after spontaneous release for 15 min at room temperature, in TTX and 2 mM calcium and after unquenching all vesicles with Nh4Cl. Scale bar = 5 μm. Values are shown as mean ± SEM.
Figure 7
Figure 7. The spontaneous pool of vesicles corresponds to the resting pool
(a) The graph shows a plot of the change in fluorescence (normalized to the Recycling pool) following different treatments. After the entire recycling pool has been mobilized with a saturating stimulus of 900 APs at 20 Hz, neurons are either treated immediately with Nh4Cl (black trace, n = 6 cells) or allowed to release spontaneously for 15 min in TTX, at room temperature and subsequently treated with Nh4Cl (red trace, n = 7 cells). The diagram below gives a temporal representation of the experimental protocol. (b) Graph showing a plot of the change in fluorescence amplitude measured with sypHy for evoked release at individual synapses (black squares) as a function of the change in fluorescence for spontaneous release. The doted black line represents the best linear fit to the data (correlation coefficient = 0.62, p < 0.05). Values are shown as mean ± SEM.

Comment in

  • Pool rules.
    Sullivan JM. Sullivan JM. Nat Neurosci. 2009 Jun;12(6):671-3. doi: 10.1038/nn0609-671. Nat Neurosci. 2009. PMID: 19471261 No abstract available.

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References

    1. Katz B. The release of neural transmitter substances. 1969
    1. Sabatini BL, Regehr WG. Timing of synaptic transmission. Annu Rev Physiol. 1999;61:521–42. - PubMed
    1. Murthy VN, Stevens CF. Reversal of synaptic vesicle docking at central synapses. Nat Neurosci. 1999;2:503–7. - PubMed
    1. Geppert M, et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell. 1994;79:717–27. - PubMed
    1. Sara Y, Virmani T, Deak F, Liu X, Kavalali ET. An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission. Neuron. 2005;45:563–73. - PubMed

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