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  1. Evaluation Summary:

    Using an elegant combination of cutting-edge techniques, the authors show that in the neuromuscular junction of the nematode C. elegans two different classes of voltage-activated calcium channels differentially trigger exocytosis of distinct pools of synaptic vesicles, one docked to the active zone and a second one localized more distant from the active zone. These findings will be of broad interest to neuroscientists interested in the mechanisms of calcium-mediated release of neurotransmitters at chemical synapses.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    In this manuscript, the authors use C. elegans as a model system to show that calcium-dependent exocytosis of synaptic vesicles is differentially coupled to two different types of calcium channels. The authors take advantage of the fact that each major calcium channel family is represented by only a single gene in C. elegans, with CaV1 corresponding to L-type, CaV2 to P/Q-type, and CaV3 to T-type calcium channels, respectively Moreover, C. elegans contains only a single ryanodine-receptor channel that is responsible for releasing calcium from intracellular stores. While it is well established that CaV2 (as in other species) is mainly responsible for exocytotic transmitter release, the role of the other channels is not clear. Here the authors use smart genetic approaches involving tissue-specific deletion of individual channels and combinations of double mutants to document that CaV1 activity is responsible for the exocytosis of a distinct class of synaptic vesicles that is farther away from the active zone, couples to a distinct short form of Unc13, and that cooperates with RyR, with the release-relevant calcium release probably mainly being derived from intracellular stores.

    With the caveat that this reviewer is not an expert in C.elegans, I consider this data-rich manuscript excellent, adding important information to the role of N-type calcium channels in transmission at the neuromuscular junction in C. elegans. As far as I can judge, the data are of high quality, and even the rather tricky spatial resolution of the adjacent release sites and the selective association of RyR with CaV1 and the short form of UNC13 using superresolution fluorescence microscopy is convincing. The manuscript is well written, and the data are succinctly discussed. As discussed by the authors it remains unclear whether such a mechanism also occurs in mammalian synapses, e.g. synapses in which exocytosis is also triggered by graded potential changes rather than by action potentials.

  3. Reviewer #2 (Public Review):

    The authors combine electrophysiology, flash freeze EM analysis of synaptic vesicle (SV) fusion, and super-resolution imaging to provide a detailed description of how CaV2 (N-type) and CaV1 (L-type) calcium channels promote neurotransmitter release at C. elegans neuromuscular junctions (NMJs). Their data suggest that SV fusions near the center of active zones are mediated by CaV2, which is highly enriched at the center of AZs. By contrast, the fusion of laterally displaced SVs (100-600 nm from the AZ center) is promoted by CaV1 and Ryanodine Receptors (RYRs) both of which are localized in a dispersed manner across the extent of the AZ. Analysis of spontaneous post-synaptic currents (PSCs) suggests that CaV2 mediated events are primarily single quantal release events, while multiquantal release events are more enriched among CaV1/RYR mediated events. Super-resolution imaging of the SV priming protein UNC-13 suggests that the short isoform (UNC-13S) is co-localized with CaV1 and RYR while the long UNC-13L isoform (inferred from the difference between total UNC-13 and UNC-13S) is tightly coupled spatially to CaV2, at AZ centers. These results significantly extend prior studies that suggested that CaV2, CaV1, RYR, UNC-13L, and UNC-13S all contribute to release at this synapse but lacked the spatial resolution to distinguish proximal and distal SV pools. Overall, these studies provide a detailed, unprecedented description of how CaV2 and CaV1 function together to promote the release of different sub-populations of SVs within a single synapse. For these reasons, this study will be of great interest to a broad group of molecular and cellular neurobiologists.

  4. Reviewer #3 (Public Review):

    In this manuscript, Jorgensen and colleagues elegantly used cutting-edge technologies to understand how different Ca entries lead to two different types of presynaptic release. They demonstrated that at the worm neuromuscular junctions two different classes of voltage-gated calcium channels, CaV2 and CaV1, mediate the release of distinct pools of synaptic vesicles. CaV2 channels are concentrated in densely packed clusters near the molecularly and EM-defined active zone structures. This type of release is dependent on synaptic vesicle priming protein UNC-13L. By contrast, they found that CaV1 channels are dispersed in synaptic varicosity and are coupled to internal calcium stores via the ryanodine receptor. CaV1 and ryanodine receptors mediate the fusion of vesicles docked broadly in synaptic varicosity and are colocalized with the vesicle priming protein UNC-13S.

    The authors were able to direct their hypotheses because they have established powerful experimental methods such as rapid freezing EM coupled with neuronal stimulation. They used genetic null mutants for most of their experiments. They created endogenously labeled proteins to test the localization of proteins in live preparations. They used a combination and electrophysiological and behavioral assays. Since they worked with a system that has a small number of synaptic connections, they can reliably study the same set of synapses. The rigor of these experiments is extremely high.

    The comprehensive approaches and the clear-cut results made this manuscript easily the top two or three papers I have read in the last couple of years of any journals.