Diversity of excitatory release sites
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Evaluation Summary:
The authors study how individual synapses can compute information by tuning the properties of the individual components that drive synaptic communication between neurons. Using cutting edge physiology and morphology they show that the reliability of synaptic communication depends not only on how many units drive synaptic communication, but also the authors suggest that individual units vary in their quantitative molecular composition.
(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 #2 agreed to share their name with the authors.)
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Abstract
The molecular mechanisms underlying the diversity of cortical glutamatergic synapses is still only partially understood. Here, we tested the hypothesis that presynaptic active zones (AZs) are constructed from molecularly uniform, independent release sites (RSs), the number of which scales linearly with the AZ size. Paired recordings between hippocampal CA1 pyramidal cells and fast-spiking interneurons followed by quantal analysis demonstrate large variability in the number of RSs ( N ) at these connections. High resolution molecular analysis of functionally characterized synapses reveals highly variable Munc13-1 content of AZs that possess the same N . Replica immunolabeling also shows a 3-fold variability in the Munc13-1 content of AZs of identical size. Munc13-1 is clustered within the AZs; cluster size and density are also variable. Our results provide evidence for quantitative molecular heterogeneity of RSs and support a model in which the AZ is built up from variable numbers of molecularly heterogeneous, but independent RSs.
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Reviewer #3 (Public Review):
Using a combination of powerful approaches authors demonstrate large variability in the number of release sites at hippocampal excitatory synapses onto fast spiking interneurons in slices. High resolution studies of individual synapses showed highly variable amounts of Munc13-1within the AZs that have the same number of release sites. The authors further revealed a synapse size-independent variability in the number of Munc13-1 clusters per AZ and in the Munc13-1 content of individual clusters. There results support the presence of multiple independent release sites and provide insight into molecular heterogeneity of release sites.
This is a high quality study using most advanced techniques available to study molecular determinants of AZ organization. In addition to some technical issues, my main concern is …
Reviewer #3 (Public Review):
Using a combination of powerful approaches authors demonstrate large variability in the number of release sites at hippocampal excitatory synapses onto fast spiking interneurons in slices. High resolution studies of individual synapses showed highly variable amounts of Munc13-1within the AZs that have the same number of release sites. The authors further revealed a synapse size-independent variability in the number of Munc13-1 clusters per AZ and in the Munc13-1 content of individual clusters. There results support the presence of multiple independent release sites and provide insight into molecular heterogeneity of release sites.
This is a high quality study using most advanced techniques available to study molecular determinants of AZ organization. In addition to some technical issues, my main concern is conceptual: this work, although of very good quality overall, is rather incremental because it largely confirms several previous studies showing a large variability in the number of release sites per AZ in small central synapses, the association between Munc-13 and release site properties, and variability in Munc-13 content. Surprisingly only one of the three of these previous studies have been cited or discussed. My second concern is that the paper could be written more clearly - there are multiple terms used to refer to the same concepts making it difficult to follow and there is some conceptual logical fallacy in the way the results are discussed.
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Reviewer #2 (Public Review):
Karlocai et al addresses a prevailing concept of synapse diversity, asking whether diversity of release probability is caused by varying number of release sites and/or the properties of individual release sites. In other words, are there functionally uniform release sites (RS) that scale in numbers with the size of the AZ and thus regulate release probability (Pv), or are, in addition, RS may be heterogeneous in composition and function. Performing quantal analysis 2.0 by combining ephys from pyramidal-to-parv interneurons in hippocampus with quantitative anatomy of a presynaptic key transducer, Munc13, they define N, Pv and Q and compare it to the numbers of munc13 clusters and densities. As expected from previous studies, RS numbers covary with the size of the AZ, but the amounts of Munc13-1 are highly …
Reviewer #2 (Public Review):
Karlocai et al addresses a prevailing concept of synapse diversity, asking whether diversity of release probability is caused by varying number of release sites and/or the properties of individual release sites. In other words, are there functionally uniform release sites (RS) that scale in numbers with the size of the AZ and thus regulate release probability (Pv), or are, in addition, RS may be heterogeneous in composition and function. Performing quantal analysis 2.0 by combining ephys from pyramidal-to-parv interneurons in hippocampus with quantitative anatomy of a presynaptic key transducer, Munc13, they define N, Pv and Q and compare it to the numbers of munc13 clusters and densities. As expected from previous studies, RS numbers covary with the size of the AZ, but the amounts of Munc13-1 are highly variable at individual RSs, providing a possible additional source of Pv variability.
Overall the quality of data is just superb, and the conclusion are well supported by the data as sufficient electrophysiological experiments were performed, and importantly also correlated with multiple, highly quantitative microscopy techniques. Only very few labs can do this at this level.
The findings carry enough impact as they negate the hypothesis that RS are made out of predefined release sites. Also, the finding that the post synapse as defined by PSD95 labeling was much less variable, indicates that pre- and postsynaptic makes do not necessarily correlate, arguing somewhat against the transsynaptic nano column concept as a main organizing principles. Thus, pre- and post-synapses are only loosely linked in their composition and function.
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Reviewer #1 (Public Review):
The authors address the broad question of what is responsible for the large diversity of presynaptic function at synapses arising from a single type of neuron. They use a variety of sophisticated and complementary approaches to address the functional and molecular heterogeneity of hippocampal pyramidal cell to fast-spiking interneuron synapses. The rigorous functional and molecular analysis is clearly described and compelling. The conclusions are consistent with the current view that each presynaptic active zone contains a variable number of release sites, and this variability makes a substantial contribution to the heterogeneity in postsynaptic response amplitude at unitary synaptic connection. Using state-of-the-art imaging approaches, the authors report variability in the content of Munc13-1, a core …
Reviewer #1 (Public Review):
The authors address the broad question of what is responsible for the large diversity of presynaptic function at synapses arising from a single type of neuron. They use a variety of sophisticated and complementary approaches to address the functional and molecular heterogeneity of hippocampal pyramidal cell to fast-spiking interneuron synapses. The rigorous functional and molecular analysis is clearly described and compelling. The conclusions are consistent with the current view that each presynaptic active zone contains a variable number of release sites, and this variability makes a substantial contribution to the heterogeneity in postsynaptic response amplitude at unitary synaptic connection. Using state-of-the-art imaging approaches, the authors report variability in the content of Munc13-1, a core component of release sites, between release sites. Although these results and conclusions are well-supported, the functional significance of Munc13-1 variability at release sites is unclear.
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Evaluation Summary:
The authors study how individual synapses can compute information by tuning the properties of the individual components that drive synaptic communication between neurons. Using cutting edge physiology and morphology they show that the reliability of synaptic communication depends not only on how many units drive synaptic communication, but also the authors suggest that individual units vary in their quantitative molecular composition.
(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 #2 agreed to share their name with the authors.)
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