title: Complexin Mutants Reveal Partial Segregation between Recycling Pathways That Drive Evoked and Spontaneous Neurotransmission.
abstract: 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, 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 that 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 electron microscopy analysis indicated that spontaneous recycling preferentially involves synaptic vesicles in the vicinity of AZs, whereas vesicles recycled following evoked release involve a larger intraterminal pool. Together, these results suggest that spontaneous and evoked vesicles use separable recycling pathways and then partially intermix during subsequent rounds of fusion.
  
  
  
  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.
authors.affiliation: Neuroscience Department, Universidad Central del Caribe, Bayamon, Puerto Rico 00960, and.
authors.forename: Nadezhda
authors.identifier: 0000-0002-7714-4884
authors.initials: N
authors.lastname: Sabeva
authors.affiliation: Picower Institute for Learning and Memory, Department of Biology, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
authors.forename: Richard W
authors.identifier: 0000-0002-8189-8785
authors.initials: RW
authors.lastname: Cho
authors.affiliation: Department of Neurology and.
authors.forename: Alexander
authors.identifier: 0000-0001-7566-3923
authors.initials: A
authors.lastname: Vasin
authors.affiliation: Neuroscience Department, Universidad Central del Caribe, Bayamon, Puerto Rico 00960, and.
authors.forename: Agustin
authors.initials: A
authors.lastname: Gonzalez
authors.affiliation: Picower Institute for Learning and Memory, Department of Biology, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
authors.forename: J Troy
authors.identifier: 0000-0001-5576-2887
authors.initials: JT
authors.lastname: Littleton
authors.affiliation: Department of Neurology and mbykhovs@med.wayne.edu.|Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan 48201.|Neuroscience Department, Universidad Central del Caribe, Bayamon, Puerto Rico 00960, and.
authors.forename: Maria
authors.initials: M
authors.lastname: Bykhovskaia
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000172:deficiency* / Q000235:genetics; D000818:Animals; D029721:Drosophila Proteins / Q000172:deficiency* / Q000235:genetics; D004331:Drosophila melanogaster; D005089:Exocytosis / Q000502:physiology*; D005260:Female; D008297:Male; D009154:Mutation / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000172:deficiency* / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology*; D009435:Synaptic Transmission / Q000502:physiology*; D013572:Synaptic Vesicles / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D029721:Drosophila Proteins; D009419:Nerve Tissue Proteins; C000605369:cpx protein, Drosophila
keywords: Drosophila; FM1–43; electron microscopy; endocytosis; exocytosis; synaptic