PONE-D-20-21322

Lactate induces synapse-specific potentiation on CA3 pyramidal cells of rat hippocampus

PLOS ONE

Dear Dr. Galvan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Your manuscript has been reviewed by two independent experts whose comments can be found below. Both experts found your study to be novel, well conducted and merits to be published but raised some concerns. I do agree with their comments notably regarding the necessity to discuss the mechanisms beyond the compartimentalisation of the effects of lactate on synaptic transmission and on cell excitability. I also agree that you may consider to use non parametric tests for your analysis rather than Student t test when size of samples is too small.

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Jean-Pierre Mothet, Ph.D

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a very nice study that examines the effects of lactate on synaptic efficacy and intrinsic excitability. Using electrophysiological techniques on acute slices of rat hippocampus, the authors convincingly show that application of lactate persistently enhances synaptic transmission at recurrent synapses but not at mossy fiber synapses in CA3 pyramidal neurons. Lactate-dependent LTP at recurrent synapses requires NMDA receptor activation, postsynaptic calcium and is sensitive to cholera toxin. The lactate induces in parallel an increase in cell excitability that is surprisingly visible on recurrent inputs but not on mossy-fiber inputs.

The question raised here is of importance, the data are clear and the paper is well written. I have however a few questions and remarks.

Excitability change illustrated in Figure 5H-J: Is it an acute effect of lactate or a long-term effect measured in parallel of synaptic transmission? Please provide details in the Methods & Results sections.

How the authors can reconcile the fact that lactate induces an increase in intrinsic excitability measured by current injection in the soma and the differential effect on recurrent and mossy-fiber inputs? A change in excitability that is global should affect equally all inputs and especially inputs located near the cell body (i.e. MF inputs). A selective change in EPSP-spike coupling can be seen only in specific conditions (see for instance Campanac et al. J Neurosci 2008).

Although, it is mentioned in the abstract, the effect of lactate on intrinsic excitability is not discussed. Please add a brief paragraph on this after clarification of the synapse specific change in excitability with at least a reference to a recent review.

Statistics: Student t-test should not be used for small samples. Please use non parametric tests (Mann-Whitney and Wilcoxon tests).

Reviewer #2: In their paper “Lactate induces synapse-specific potentiation on CA3 pyramidal cells of rat Hippocampus”, Herrera-Lopez et al. report input source-dependent potentiation of synaptic transmission onto hippocampal C3 pyramidal cells by lactate, and suggest that this effect is mediated by a Gs-protein-coupled lactate receptor. Synaptic potentiation applies only to recurrent C3 synapses but not to mossy fiber synapses. In addition, lactate causes transient synaptic depression presumably via a metabolism-related effect. To note, a physiologically relevant range of lactate concentrations was tested. This is very exciting new data and substantiated by electrophysiological and pharmacological approaches in mature rodent tissue.

The text is well written, with very few typographical and syntax errors (listed under specific comments below). Description of methods is mostly transparent, apart from some aspects of the electrical stimulation protocol which I would ask to clarify in more detail (see specific comments). With a few exceptions (see specific comments), the figures are well designed and clearly described in results section as well as legends.

My main concern is that the mechanistical interpretation of the differences between lactate effects on RC and MF transmission requires further development. The Discussion and Figure 8 appear to ascribe the lactate-induced synaptic potentiation in RC, but not MF, synapses to postsynaptic changes. However, since the postsynaptic cell is identical (a CA3 PC), the logic isn’t obvious, unless the authors would want to explain this difference by compartmentalisation and local signalling of the G-protein-coupled lactate receptor? Their reasoning should be explained in more detail in the Discussion to include lactate actions on MF as well as RC synapses, and this should be reflected by Figure 8.

Specific comments:

Line 92: … and then kept for 90 min…

Line 123 etc – Stimulation protocol unclear: What does “70 ms at 0.06 Hz” mean and how do these parameters relate to the “tandem analysis” introduced in line 161 as “…acquired with an ISI of 1 s and stimulation frequency of 0.16 Hz”? Also see legend to Fig 1.

The time course plots in the Figures (e.g. 1B) suggest a stimulation frequency of ~0.02 Hz for each pathway. If “tandem” means alternating stimulation between MF and RC, I would expect them to be applied in equal ~25 sec intervals, in order for activation of one pathway not to bias the response of the other. Please clarify in the Methods section what exactly was done.

Also, it is not explained how CV^-2 (first appearance line 234) was determined and that should be done in order to ensure accessibility to a wider readership.

Lastly, one assumes that what is reported are normalised EPSC amplitudes but that isn’t explicitly stated in the manuscript.

Line 137 – Drugs: Lactate not mentioned; please also state pH of stock solution.

General: Inconsistent use of abbreviation for pyramidal cells, suggest to introduce at first instance in main text and then use throughout.

Lines 209,211: Because… Because (suggest to replace one with “Since”)

Lines 213-14: “…capable of potentiating the synaptic…”

Line 216: “…EPSC to a similar extent…”

Fig 2D: LTP label – why “LTP”? this should probably be representing “synaptic potentiation”.

Line 314: “… of CA3 PC have a predominant NMDAR component involved in RC long-term…”

Line 361-362: “…of lactate. Although pyruvate (2 mM) did not induce RC EPSC potentiation, it did

increase, in a transient manner, the RC…”

Lines 400 etc: Legend to Figure 4B is missing (and much needed as the results text doesn’t make it very clear what was done here) – the text to B) seems to refer to panel C, etc.

Fig 5B: LTP label – should say 3,5-DHBA? In this case, the figure represents “synaptic potentiation” but not LTP (as in Figs 3, 7)?

Line 646: “…Lastly, although our occlusion experiments…”

Lines 724 – 729: Sentence incomplete

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Reviewer #1: No

Reviewer #2: No

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Lactate induces synapse-specific potentiation on CA3 pyramidal cells of rat hippocampus

PONE-D-20-21322R1

Dear Dr. Galvan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Jean-Pierre Mothet, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper has been adequately revised (statiistics use non-parametric tests, and previous concerns have been dissipated). I have no further comment.

Reviewer #2: I am satisfied with revisions the authors have undertaken, and appreciate the efforts gone into more detailed interpretation of the differential effects of lactate on RC and MF synaptic transmission, including the improvements in Fig 8. The legend would still benefit from some cuts and rephrasing. For example, for a title “Proposed mechanisms of lactate actions at the excitatory synapses of the CA3 pyramidal cells” may be more precise; “Schematic representation.” is unnecessary; the explanation of Gs-protein activation (“…induces a conformational change …”) seems too extensive; pyruvate does not “synthesize ATP”, nor adenosine.

As the authors acknowledge in their revised manuscript, a number of steps in their model will require further clarification through future work. I suggest these would include the currently unexplained mechanism of the proposed post-synaptic release of ATP. In fact, local astrocytes as prime sources of extracellular ATP and lactate signalling may well be involved in the synaptic potentiation processes described in this paper.

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Reviewer #1: No

Reviewer #2: No