Review Reports - Neurotransmitter Release Site Replenishment and Presynaptic Plasticity

Round 1

Reviewer 1 Report

Review: Neurotransmitter release site replenishment and presynaptic plasticity

The review covers the main and recent topics in synaptic transmission on a focus of the presynaptic site. The focus on Ca2+ dynamics was helpful in pulling together the timing of vesicle fusion and Ca2+ regulation as well as the role of CAST. The relation with various model preparations was helpful, such as bruchpilot in Drosophila with CAST for mammalian preparations.

Minor:

Line 198: "Although its role….". Maybe state what "its" means just to be clear

Line 210: Generally, the RRP is referred to as " readily releasable pool" not the "release-ready" just seems odd not to use the standard terminology. (Kaeser, P.S.;Regehr, W.G. The readily releasable pool of synaptic vesicles. Curr Opin Neurobiol 2017,43: 63-70.)

Line234 other places as well:

Just using the word "AP" in the way the author uses it .... implies "APs" are occurring so there is no need to state AP "firing". Maybe just state AP. "That a single AP is not sufficient to…."

Line 241: "Following APs at 10 Hz….." would read better.

Or "Following APs occurring at 10 Hz…

The author may like to know about these references

Low freq depression..................Silverman-Gavrila LB, Praver M, Mykles DL, Charlton MP. Calcium, calpain, and calcineurin in low-frequency depression of transmitter release. J Neurosci. 2013 Jan 30;33(5):1975-90. doi: 10.1523/JNEUROSCI.3092-12.2013. PMID: 23365236; PMCID: PMC6619111
The review cited covers a lot (Rizzoli, S.O.;Betz, W.J. Synaptic vesicle pools. Nat Rev Neurosci2005, 6(1), 57-69.) But this review in 2014 covers some of the topics in the current manuscript which is worth citing.
considering vesicles may recycle empty, can one be sure the vesicles are really inhibited from fusion when measuring by quantal responses?

Author Response

Responses to reviewer 1

Thank you very much for your valuable comments that help my poor English writing and knowledge of published papers. My corrections pointed by reviewer 1 are highlighted with green color in the text.

Line 198: "Although its role….". Maybe state what "its" means just to be clear => “Its role” is changed to “role of myosin”

Line234 other places as well:

Just using the word "AP" in the way the author uses it .... implies "APs" are occurring so there is no need to state AP "firing". Maybe just state AP. "That a single AP is not sufficient to…." => "firing" is removed throughout of the text, indicated with green highlight.

Line 241: "Following APs at 10 Hz….." would read better. => Text was changed to “Following APs at 10 Hz”

The author may like to know about these references

Low freq depression..................Silverman-Gavrila LB, Praver M, Mykles DL, Charlton MP. Calcium, calpain, and calcineurin in low-frequency depression of transmitter release. J Neurosci. 2013 Jan 30;33(5):1975-90. doi: 10.1523/JNEUROSCI.3092-12.2013. PMID: 23365236; PMCID: PMC6619111

=>line 406: New description is added.

Synaptic depression is often related to Ca²⁺-dependent phosphorylation of synaptic proteins. Long-term depression in cerebellar [110] and hippocampal synapses [111], for example, involve presynaptic and postsynaptic activity of the Ca²⁺/CaM-activated protein phosphatase. In addition, low-frequency depression of transmitter release caused by stimulation at 0.2 Hz, at crayfish phasic synapses, requires presynaptic phosphatase calcineurin activated by Ca²⁺/calpain [112].

The review cited covers a lot (Rizzoli, S.O.; Betz, W.J. Synaptic vesicle pools. Nat Rev Neurosci 2005, 6(1), 57-69.) But this review in 2014 covers some of the topics in the current manuscript which is worth citing.

=> Rizzoli, S.O., Synaptic vesicle recycling: steps and principles. Embo j 2014, 33(8), 788-822. This review is added in the list and cited as [82]

considering vesicles may recycle empty, can one be sure the vesicles are really inhibited from fusion when measuring by quantal responses?

=> line 331: New description is added. It should be noted that a single AP-triggered quantal release at mammalian end-plate measured in low Ca²⁺ or high Mg²⁺showed single and multiple vesicles release and less failure [94], suggesting membrane retrieval can be achieved even in low level of Ca²⁺ rise in the AZ.

In addition, introduction and several important findings pointed by other reviewers are added. 1) line 45-54, introduction, 2) line 100-106, the “kiss-and-run” event, 3) line 108-119, change in Ca²⁺ channel distribution in developing and mature calyx of Held synapses, 4) line 120-127, capture of synaptic vesicle by Ca_V2.1 and Ca_V2.2 channels.

Reviewer 2 Report

This is a highly competent review article by a leader in the synaptic transmission field. The work is well organized, and nicely illustrated, and will make a useful contribution to the field. I have two minor comments, as well as one slightly more substantial consideration.

Slightly major:

There is a series of papers from Elise Stanley's lab in Frontiers in Cellular Neuroscience (2013, 2014,2017,2018) that deals with capture and tethering of of synaptic vesicle. This work was ignored her even though it might have been relevant to section 4. This should have been included one way or another (even if you do not agree with it). Also, work on calyx of Held is mentioned, but some of the seminal work from Dr. Lu-yang Wang's group is not cited (for example Fekete et al, Nat Communications). I realize that the topic is not as much about vesicle release as about replenishment, but synaptic architecture vis a vis calcium channels is discussed here and so the author may wish to include the above works.

Minor:

In the section on calmodulin, "robe" should be "lobe"

In contrast "Endocytosis regulates release-ready SVs restoration" is incorrect and should be "SV restoration" or alternatively, "restoration of SVs" - this type of error happens many times throughout the text. So every time you use "SVs" as an abbreviation, please make sure that it is correctly used

Author Response

Responses to reviewer 2

Thank you very much for your valuable comments that help my poor English writing and knowledge of published papers. My corrections listed by reviewer 2 are highlighted with blue color in the text.

Slightly major:

=> Works on calyx of Held synapse of Thakahashi’s goup and Lu-yang Wang’s group and series of papers from Stanley’s lab were summarized in the new subsection 3.1. 3.1. SV docking and Ca channel cluster in AZ

AZ serves as a platform for linking SVs to Ca²⁺ channels through proteins complex (Figure 2). The distance between docked SVs and Ca channels that determinates transmitter release probability differs with AZ architecture. In developing calyx of Held from P7 to P14 the distance between SV and Ca²⁺ channel cluster decrease from 30 nm to 20 nm, and the number of Ca_V2.1 channel per cluster and the cluster area increase with development, however, the density of Ca_V2.1 channel remains similar [24]. The mature calyx of Held synapse has numbers of bouton-like swellings on stalks of the nerve terminals that show lower release probability than that of stalks. Wong and colleagues, measuring distance of fluorescently tagged-Ca²⁺ and SVs coupling using Ca²⁺ chelator, explored that larger clusters of Ca²⁺ channels with tighter coupling distance to SVs elevate release probability in stalks, while smaller clusters with looser coupling distance lower release probability in swellings [25].

As describing in next 3.2 section, Ca²⁺ channels are installed in the AZ membrane by AZ protein(s) which mediates linkage of docked SVs and Ca²⁺ channels at the release site. Stanley proposes that a single Ca²⁺ channel domain gates SV fusion at fast synapse [26]. She and co-workers developed an in vitro SV pull-down assay [27] and presented evidence that Ca_V2.1 channel or the mid-region of its C-terminal captures an SV [28], and Ca_V2.2 channels or the distal third of its C-terminal [29] capture an SV as far as 100 nm from the AZ region [30, 31]. They hypothesized that one or more additional linker molecule(s) lock the captured SV within its Ca²⁺ sensor trap Ca²⁺ at the channel mouth [31].

Minor:

In the section on calmodulin, "robe" should be "lobe" => corrected.

The author uses SV as an abbreviation for synaptic vesicle. Here, plural is used incorrectly throughout the manucript - for example, if the authors want to say replenishment of synaptic vesicles, then "SVs" should be used. In contrast "Endocytosis regulates release-ready SVs restoration" is incorrect and should be "SV restoration" or alternatively, "restoration of SVs" - this type of error happens many times throughout the text. So every time you use "SVs" as an abbreviation, please make sure that it is correctly used. => carefully corrected throughout of the text.

In addition, introduction and several important findings pointed by other reviewers are added. 1) line 45-54, introduction, 2) line 100-106, the “kiss-and-run” event, 3) line 331-333, the “quantal release” event, 4) line 406-410, presynaptic depression

Reviewer 3 Report

The present review is well written and clear. Several very accurate details are reported by the author about the synaptic vesicles machinery. Few points require attention in my opinion:

Introduction section is not well focused, and can better explain the literature concerning the main arguments reported in the rest of the paper

Figures are too detailed reporting several plots and graphs. I think this is redundant for a review and make difficult sometime to follow the text

Author report several data about synapse model based on sympathetic superior cervical ganglion neurons. Is some case these data seems overrepresented with respect the rest of the results.

Pg 2 line 46, reference reported seems wrong

Author Response

Responses to reviewer 3

Thank you very much for your valuable comments. My corrections pointed by reviewer 3 are highlighted with pink color in the text.

Introduction section is not well focused, and can better explain the literature concerning the main arguments reported in the rest of the paper => Thank you for your pointing out. I added description explaining it.

This review introduces, at first, recent findings on temporal regulation of SV states within 100 ms of a single AP, and then our findings on millisecond Ca²⁺ dynamics dependent transmitter release site replenishment with release-ready SVs that involves multiple protein cascades, such as phosphorylation of zone proteins, activation of myosin motors and that of key proteins linking exocytosis and endocytosis. These protein reactions controlled by Ca²⁺ sensors underlie the presynaptic short-term plasticity. Furthermore, I would introduce an important regulation of Ca²⁺ elevation in the AZ. After a single AP, Ca²⁺ channel opening is temporally fine-controlled by the AP-induced Ca²⁺ dynamics via Ca²⁺ sensors bound to the Ca²⁺ channel. Regulation of Ca²⁺ elevation significantly controls state and replenishment of SVs and contributes presynaptic plasticity.

Figures are too detailed reporting several plots and graphs. I think this is redundant for a review and make difficult sometime to follow the text => Thank you for your pointing out. I divided Figure 2 to Figure 2 and 3, and Figure 3 to Figure 4 and Figure 5, hoping easier understanding of figure contents.

Author report several data about synapse model based on sympathetic superior cervical ganglion neurons. Is some case these data seems overrepresented with respect the rest of the results. => Several important findings pointed by other reviewers are added. 1) line 100-106, the “kiss-and-run” event, 2) line 108-119, change in Ca²⁺ channel distribution in developing and mature calyx of Held synapses, 3) line 120-127, capture of synaptic vesicle by Ca_V2.1 and Ca_V2.2 channels, 4) line 331-333, the “quantal release” event, 5) line 406-410, presynaptic depression.

Pg 2 line 46, reference reported seems wrong => corrected to Ma, H.; Mochida, S. A cholinergic model synapse to elucidate protein function at presynaptic terminals. Neurosci Res 2007, 57(4), 491-8.