PT  - JOURNAL ARTICLE
AU  - Nadezhda Sabeva
AU  - Richard W. Cho
AU  - Alexander Vasin
AU  - Agustin Gonzalez
AU  - J. Troy Littleton
AU  - Maria Bykhovskaia
TI  - Complexin Mutants Reveal Partial Segregation Between Recycling Pathways that Drive Evoked and Spontaneous Neurotransmission
AID  - 10.1523/JNEUROSCI.1854-16.2016
DP  - 2016 Dec 02
TA  - The Journal of Neuroscience
PG  - 1854-16
4099  - http://www.jneurosci.org/content/early/2016/12/02/JNEUROSCI.1854-16.2016.short
4100  - http://www.jneurosci.org/content/early/2016/12/02/JNEUROSCI.1854-16.2016.full
AB  - Synaptic vesicles fuse at morphological specializations in the presynaptic terminal termed active zones (AZs). Vesicle fusion can occur spontaneously or in response to an action potential. Following fusion, vesicles are retrieved and recycled within nerve terminals. It is still unclear whether vesicles that fuse spontaneously or following evoked release share similar recycling mechanisms. Genetic deletion of the SNARE-binding protein complexin dramatically increases spontaneous fusion, with the protein serving as the synaptic vesicle fusion clamp at Drosophila synapses. We examined synaptic vesicle recycling pathways at complexin null neuromuscular junctions, where spontaneous release is dramatically enhanced. We combined loading of the lipophilic dye FM1-43 with photoconversion, electron microscopy (EM), and electrophysiology to monitor evoked and spontaneous recycling vesicle pools. We found that the total number of recycling vesicles was equal to those retrieved through spontaneous and evoked pools, suggesting retrieval following fusion is partially segregated for spontaneous and evoked release. In addition, the kinetics of FM1-43 destaining and synaptic depression measured in the presence of the vesicle refilling blocker bafilomycin indicated that spontaneous and evoked recycling pools partially intermix during the release process. Finally, FM1-43 photoconversion combined with EM analysis indicated spontaneous recycling preferentially involves synaptic vesicles in the vicinity of AZs, while vesicles recycled following evoked release involve a larger intra-terminal pool. Together, these results suggest that spontaneous and evoked vesicles use separable recycling pathways and then partially intermix during subsequent rounds of fusion.Significance Statement:Neurotransmitter release involves fusion of synaptic vesicles with the plasma membrane in response to an action potential, or spontaneously in the absence of stimulation. Upon fusion, vesicles are retrieved and recycled, and it is unclear whether recycling pathways for evoked and spontaneous vesicles are segregated after fusion. We addressed this question by taking advantage of preparations lacking the synaptic protein complexin, which have elevated spontaneous release that enables reliable tracking of the spontaneous recycling pool. Our results suggest that spontaneous and evoked recycling pathways are segregated during the retrieval process, but can partially intermix during stimulation.