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

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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Figure 1: Visualizing vesicles released by membrane depolarization using biosyn.
Figure 2: Biosyn can be used as a reliable tool to assess evoked vesicle fusion.
Figure 3: Visualizing vesicles released spontaneously with biosyn.
Figure 4: There are two distinct pools of vesicle with different release modes: spontaneous and evoked.
Figure 5: Quantification of fluorescence intensity for different conditions.
Figure 6: Measuring vesicle pools with sypHy.
Figure 7: The spontaneous pool of vesicles corresponds to the resting pool.

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Acknowledgements

We would like to thank O. Burrone for kindly donating the modified BirA expression construct and for helpful advice and discussion on experimental protocols. We would also like to thank I. Thompson and the members of the Thompson and Burrone laboratories for helpful discussions and suggestions. We would finally like to thank V.N. Murthy for his input in the initial stages of this project and L. Lagnado, Q. Ch'ng, M. Grubb and L. Andreae for critical reading of the manuscript. This work was supported by a Medical Research Council project grant and a Wellcome Trust project grant to J.B.

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Correspondence to Juan Burrone.

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Fredj, N., Burrone, J. A resting pool of vesicles is responsible for spontaneous vesicle fusion at the synapse. Nat Neurosci 12, 751–758 (2009). https://doi.org/10.1038/nn.2317

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