Calcium dependence of neurotransmitter release at a high fidelity synapse
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Evaluation Summary:
The authors examined the Ca-dependence of exocytosis at cerebellar mossy fiber boutons using eectrophysiology, Ca imaging, Ca uncaging and capacitance measurements. The study reveals the presence of a high affinity Ca sensor for exocytosis, a shallow, seemingly non-saturating relationship between Ca and release or Ca and synaptic delay, a high-affinity sensory for priming of vesicles with very low (near basal) Ca levels, a late rate of release that is independent of Ca concentration (presumably due to sensor saturation), and extremely fast peak kinetics of release. This work contributes to a comparative view of synapses. These general approaches have been used at other synapses over many years by Neher and others, and they show intriguing differences among different types of synapse that are likely functionally significant. A strength of this manuscript is the masterful implementation and explanation of the techniques. The recordings at physiological temperatures, pushing-the-envelope for speed of capacitance measurements, the very careful measurement of KDs of indicators, and the unbiased testing of diverse modern-day kinetic models for release, all combine to lend the paper reliability and give it lasting value.
(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. The reviewers remained anonymous to the authors.)
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Abstract
The Ca 2+ -dependence of the priming, fusion, and replenishment of synaptic vesicles are fundamental parameters controlling neurotransmitter release and synaptic plasticity. Despite intense efforts, these important steps in the synaptic vesicles’ cycle remain poorly understood due to the technical challenge in disentangling vesicle priming, fusion, and replenishment. Here, we investigated the Ca 2+ -sensitivity of these steps at mossy fiber synapses in the rodent cerebellum, which are characterized by fast vesicle replenishment mediating high-frequency signaling. We found that the basal free Ca 2+ concentration (<200 nM) critically controls action potential-evoked release, indicating a high-affinity Ca 2+ sensor for vesicle priming. Ca 2+ uncaging experiments revealed a surprisingly shallow and non-saturating relationship between release rate and intracellular Ca 2+ concentration up to 50 μM. The rate of vesicle replenishment during sustained elevated intracellular Ca 2+ concentration exhibited little Ca 2+ -dependence. Finally, quantitative mechanistic release schemes with five Ca 2+ binding steps incorporating rapid vesicle replenishment via parallel or sequential vesicle pools could explain our data. We thus show that co-existing high- and low-affinity Ca 2+ sensors mediate priming, fusion, and replenishment of synaptic vesicles at a high-fidelity synapse.
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Evaluation Summary:
The authors examined the Ca-dependence of exocytosis at cerebellar mossy fiber boutons using eectrophysiology, Ca imaging, Ca uncaging and capacitance measurements. The study reveals the presence of a high affinity Ca sensor for exocytosis, a shallow, seemingly non-saturating relationship between Ca and release or Ca and synaptic delay, a high-affinity sensory for priming of vesicles with very low (near basal) Ca levels, a late rate of release that is independent of Ca concentration (presumably due to sensor saturation), and extremely fast peak kinetics of release. This work contributes to a comparative view of synapses. These general approaches have been used at other synapses over many years by Neher and others, and they show intriguing differences among different types of synapse that are likely functionally …
Evaluation Summary:
The authors examined the Ca-dependence of exocytosis at cerebellar mossy fiber boutons using eectrophysiology, Ca imaging, Ca uncaging and capacitance measurements. The study reveals the presence of a high affinity Ca sensor for exocytosis, a shallow, seemingly non-saturating relationship between Ca and release or Ca and synaptic delay, a high-affinity sensory for priming of vesicles with very low (near basal) Ca levels, a late rate of release that is independent of Ca concentration (presumably due to sensor saturation), and extremely fast peak kinetics of release. This work contributes to a comparative view of synapses. These general approaches have been used at other synapses over many years by Neher and others, and they show intriguing differences among different types of synapse that are likely functionally significant. A strength of this manuscript is the masterful implementation and explanation of the techniques. The recordings at physiological temperatures, pushing-the-envelope for speed of capacitance measurements, the very careful measurement of KDs of indicators, and the unbiased testing of diverse modern-day kinetic models for release, all combine to lend the paper reliability and give it lasting value.
(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. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
From a technical point of view this is clearly a very well-done study that does a good job of getting the details right and thoroughly describing the technical issues and the methods. The quantification of presynaptic calcium levels is very convincing. The study is enhanced by the ability to measure both presynaptic capacitance changes as well as postsynaptic AMPA currents. The results are interesting and important.
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Reviewer #2 (Public Review):
In this paper by Eshra et al., the authors have examined the Ca-dependence of exocytosis at cerebellar mossy fiber boutons. Electrophysiology, Ca imaging, Ca uncaging and capacitance measurements were used. The study reveals the presence of a high affinity Ca sensor for exocytosis, a shallow, seemingly non-saturating relationship between Ca and release or Ca and synaptic delay, a high-affinity sensory for priming of vesicles with very low (near basal) Ca levels, a late rate of release that is independent of Ca concentration (presumably due to sensor saturation), and extremely fast peak kinetics of release. In a way, this work contributes to a comparative view of synapses: these general approaches have been used at other synapses over many years by Neher and others, and they show intriguing differences among …
Reviewer #2 (Public Review):
In this paper by Eshra et al., the authors have examined the Ca-dependence of exocytosis at cerebellar mossy fiber boutons. Electrophysiology, Ca imaging, Ca uncaging and capacitance measurements were used. The study reveals the presence of a high affinity Ca sensor for exocytosis, a shallow, seemingly non-saturating relationship between Ca and release or Ca and synaptic delay, a high-affinity sensory for priming of vesicles with very low (near basal) Ca levels, a late rate of release that is independent of Ca concentration (presumably due to sensor saturation), and extremely fast peak kinetics of release. In a way, this work contributes to a comparative view of synapses: these general approaches have been used at other synapses over many years by Neher and others, and they show intriguing differences among different types of synapse that are likely functionally significant. A strength of the work however is in the masterful implementation and explanation of the techniques. The recordings at physiological temperatures, pushing-the-envelope for speed of capacitance measurements, the very careful measurement of KDs of indicators, and the unbiased testing of diverse modern-day kinetic models for release, all combine to lend the paper reliability and give it lasting value.
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Reviewer #3 (Public Review):
By combining patch clamp recordings at the cerebellar mossy fiber bouton/granule cell synapse with calcium uncaging and two photon Ca2+ imaging Eshra et al, directly correlate presynaptic Ca2+ levels to neurotransmitter release rates. Subsequently, they use these quantitative measurements to test three different previously published models of neurotransmitter release to demonstrate that neurotransmitter release at the cerebellar mossy fiber bouton is best described by models with 5-site Ca2+ binding steps with either parallel or sequential pools. Furthermore, they demonstrate like many other presynaptic terminals, at the cerebellar mossy fiber bouton that there are synaptic vesicles with different intrinsic Ca2+ sensitivity. The experiments are highly rigorous, and the data quality is excellent. Furthermore, …
Reviewer #3 (Public Review):
By combining patch clamp recordings at the cerebellar mossy fiber bouton/granule cell synapse with calcium uncaging and two photon Ca2+ imaging Eshra et al, directly correlate presynaptic Ca2+ levels to neurotransmitter release rates. Subsequently, they use these quantitative measurements to test three different previously published models of neurotransmitter release to demonstrate that neurotransmitter release at the cerebellar mossy fiber bouton is best described by models with 5-site Ca2+ binding steps with either parallel or sequential pools. Furthermore, they demonstrate like many other presynaptic terminals, at the cerebellar mossy fiber bouton that there are synaptic vesicles with different intrinsic Ca2+ sensitivity. The experiments are highly rigorous, and the data quality is excellent. Furthermore, the experiments are a technical tour de force as direct presynaptic recordings are technically challenging and not trivial to perform. These results are important and novel, as they add to our understanding on the presynaptic mechanisms that are used at synapses to enable high fidelity information encoding. However, this reviewer has some criticisms for the authors to consider to strengthen their story. Overall, the study is very solid with all the appropriate experimental approaches and is extremely rigorous. Previously, it has been described by Lou et al Nature 2005 that an allosteric model, not a 5-site model best describes Ca2+ sensitivity of neurotransmitter release. However, there is no data on the the Ca2+ dose response curve at Ca2+ concentrations lower than 1 µM. This is critical to know if an allosteric model can describe release at the cerebellar mossy fiber bouton.
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