Two forms of asynchronous release with distinctive spatiotemporal dynamics in central synapses
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eLife assessment
This study addresses a fundamental question about the spatiotemporal location of neurotransmitter release in a synapse with essential implications for postsynaptic signaling and neural excitability in general. The authors provide convincing evidence on non-overlapping nanometer scale organization of the two primary forms of evoked vesicle fusion (synchronous and asynchronous) in the synapse. They utilize tools for super-resolution assessment of synaptic transmission that were previously developed in their lab, in this way they help bridge earlier work based on imaging approaches that lack temporal resolution and electrophysiological results lacking spatial resolution.
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Abstract
Asynchronous release is a ubiquitous form of neurotransmitter release that persists for tens to hundreds of milliseconds after an action potential. How asynchronous release is organized and regulated at the synaptic active zone (AZ) remains debatable. Using nanoscale-precision imaging of individual release events in rat hippocampal synapses, we observed two spatially distinct subpopulations of asynchronous events, ~75% of which occurred inside the AZ and with a bias towards the AZ center, while ~25% occurred outside of the functionally defined AZ, that is, ectopically. The two asynchronous event subpopulations also differed from each other in temporal properties, with ectopic events occurring at significantly longer time intervals from synchronous events than the asynchronous events inside the AZ. Both forms of asynchronous release did not, to a large extent, utilize the same release sites as synchronous events. The two asynchronous event subpopulations also differ from synchronous events in some aspects of exo-endocytosis coupling, particularly in the contribution from the fast calcium-dependent endocytosis. These results identify two subpopulations of asynchronous release events with distinctive organization and spatiotemporal dynamics.
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eLife assessment
This study addresses a fundamental question about the spatiotemporal location of neurotransmitter release in a synapse with essential implications for postsynaptic signaling and neural excitability in general. The authors provide convincing evidence on non-overlapping nanometer scale organization of the two primary forms of evoked vesicle fusion (synchronous and asynchronous) in the synapse. They utilize tools for super-resolution assessment of synaptic transmission that were previously developed in their lab, in this way they help bridge earlier work based on imaging approaches that lack temporal resolution and electrophysiological results lacking spatial resolution.
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Reviewer #1 (Public Review):
In the manuscript Malagon et al. investigate the nano-organization of asynchronous release at glutamatergic synapses. The authors conduct near-TIRF imaging to probe the localization of synchronous and asynchronous release sites at a single synapses using vGlut-pHluorin. Recent work in the field of synaptic neurobiology has focused on investigating how different modes of neurotransmission are organized in the presynaptic bouton, however, discrepancy remains on the sub-synaptic localization of asynchronous release sites and whether these are independent from synchronous release locations. While a variety of techniques including flash-freeze EM and super resolution microscopy have been employed, the use of live imaging by Malagon et al. provides further insight at the single synapse level.
With an impressive …
Reviewer #1 (Public Review):
In the manuscript Malagon et al. investigate the nano-organization of asynchronous release at glutamatergic synapses. The authors conduct near-TIRF imaging to probe the localization of synchronous and asynchronous release sites at a single synapses using vGlut-pHluorin. Recent work in the field of synaptic neurobiology has focused on investigating how different modes of neurotransmission are organized in the presynaptic bouton, however, discrepancy remains on the sub-synaptic localization of asynchronous release sites and whether these are independent from synchronous release locations. While a variety of techniques including flash-freeze EM and super resolution microscopy have been employed, the use of live imaging by Malagon et al. provides further insight at the single synapse level.
With an impressive resolution of 27nm in live synapses the authors are able delineate synchronous and asynchronous release events within the same active zone. Furthermore, beyond the pure localization of release sites, how the vGlut-pHluorin fluorescent signal decays following fusion provides insight into distinct endocytic mechanisms. The authors delineate two populations of asynchronous events - one located within the active zone center and one ectopically outside this map (as defined by synchronous release sites). Synchronous and asynchronous demonstrate similar kinetics for the ultra-fast component of endocytosis with major differences in the fast component, which is calcium dependent for synchronous release. The authors demonstrate a consistent pattern in the localization and kinetics of release across multiple types of experiments with both EGTA and Sr2+ manipulation.
Inclusion of further analyses on already acquired data would greatly strengthen the paper, such as if single synapses preference one type of release over another. While this paper reconciles differences in the field major questions still remain; what is the mechanism for calcium independent and calcium dependent endocytosis and how does this differ between synchronous and asynchronous release. This paper sets the stage for further work probing what presynaptic machinery drives the segregation of release, what proteins mediate the differences in exocytosis-endocytosis coupling, and how the nano-organization of asynchronous release sites imparts autonomous roles for asynchronous release.
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Reviewer #2 (Public Review):
Synaptic transmission is a fundamental process of communication in the brain. How and where neurotransmitter release occurs is still an open question. This study addresses an interesting question about the spatiotemporal location of neurotransmitter release in a synapse. This has important implications for postsynaptic signaling and neural excitability in general. The work provides valuable additions to recently uncovered discrepancies in the nanometer scale organization of the two primary forms of evoked vesicle fusion (synchronous and asynchronous) in the synapse from two very different methods. Essentially demonstrating convincingly that synchronous and asynchronous release sites are unshared. The study utilizes tools of super-resolution measurements of synaptic transmission that were previously developed …
Reviewer #2 (Public Review):
Synaptic transmission is a fundamental process of communication in the brain. How and where neurotransmitter release occurs is still an open question. This study addresses an interesting question about the spatiotemporal location of neurotransmitter release in a synapse. This has important implications for postsynaptic signaling and neural excitability in general. The work provides valuable additions to recently uncovered discrepancies in the nanometer scale organization of the two primary forms of evoked vesicle fusion (synchronous and asynchronous) in the synapse from two very different methods. Essentially demonstrating convincingly that synchronous and asynchronous release sites are unshared. The study utilizes tools of super-resolution measurements of synaptic transmission that were previously developed in their lab, which help bridge the discrepancies using a convincing number of experimental paradigms. Limits in the speed of optical resolution opened a few questions of interpretation. However, this study greatly expands our knowledge of synaptic architecture and function of different forms of release. A further claim of different coupling of vesicle fusion and retrieval kinetics is made that at present seems incomplete due to temporal limitation of the super-resolution method.
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Reviewer #3 (Public Review):
This important study uses high resolution imaging of single synaptic vesicle fusion events to look at the localization of individual vesicle vGlut-pHluuorin fusion events. Using this approach, the authors were able to determine with high resolution the location of single vesicle fusion. The authors find that a significant percentage of asynchronous events occur ectopically outside the synapse, but that most still fuse within the synapse and that the fluorescent decay rates, as a proxy for vesicle endocytosis change with localization within the synapse.
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