Author response:
The following is the authors’ response to the current reviews.
We thank the editor and reviewers for their thoughtful evaluations. We would like to clarify that the revised manuscript does not make a general claim about the absence of ripple-associated synchronous population activity. Rather, we report only that the synchronous ensembles observed in our data were not associated with contralateral ripple oscillations. This distinction is clearly reflected in the revised Title, Abstract, Introduction, Results, and Discussion. We also explicitly acknowledged the methodological limitation of recording LFP from the contralateral side of the hippocampus.
To further improve clarity and prevent potential misinterpretation, we are submitting a revised version (R4) in which we:
(1) Replace the word "surprisingly" with the more neutral "Moreover";
(2) Refer to ripple events consistently as "contralateral ripples (c-ripples)";
(3)Expand the discussion of limitations inherent to contralateral LFP recordings.
Additionally, while Buzsaki et al. (2003) wrote that "These findings suggest ripples emerge locally and independently in the two hemispheres", the same study also presents data and reports that "Ripple episodes occurred simultaneously in the left and right CA1 regions" (p. 206). Our original citation was intended to reflect this nuance. Nevertheless, to avoid any potential misinterpretation, we have removed the co-occurrence statement with its associated citations in the revised (R4) manuscript.
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
For many years, there has been extensive electrophysiological research investigating the relationship between local field potential patterns and individual cell spike patterns in the hippocampus. In this study, using state-ofthe-art imaging techniques, they examined spike synchrony of hippocampal cells during locomotion and immobility states. In contrast to conventional understanding of the hippocampus, the authors demonstrated that hippocampal place cells exhibit prominent synchronous spikes locked to theta oscillations.
Strengths:
The voltage imaging used in this study is a highly novel method that allows recording not only suprathreshold-level spikes but also subthreshold-level activity. With its high frame rate, it offers time resolution comparable to electrophysiological recordings.
Comments on revisions: I have no further comments.
We thank the reviewer for constructive reviews and for recognizing the strength of our study.
Reviewer #2 (Public review):
Summary:
This study employed voltage imaging in the CA1 region of the mouse hippocampus during the exploration of a novel environment. The authors report synchronous activity, involving almost half of the imaged neurons, occurred during periods of immobility. These events did not correlate with SWRs, but instead, occurred during theta oscillations and were phased locked to the trough of theta. Moreover, pairs of neurons with high synchronization tended to display non-overlapping place fields, leading the authors to suggest these events may play a role in binding a distributed representation of the context.
Strengths:
Technically this is an impressive study, using an emerging approach that allows single cell resolution voltage imaging in animals, that while head-fixed, can move through a real environment. The paper is written clearly and suggests novel observations about population level activity in CA1.
Comments on revisions:
I have no further major requests and thank the authors for the additional data and analyses.
We thank the reviewer for recognizing the strength of our study and for appreciating the additional data and analyses we provided during the revision process.
Reviewer #3 (Public review):
Summary:
In the present manuscript, the authors use a few minutes of voltage imaging of CA1 pyramidal cells in head fixed mice running on a track while local field potential (LFPs) are recorded. The authors suggest that synchronous ensembles of neurons are differentially associated with different types of LFP patterns, theta and ripples. The experiments are flawed in that the LFP is not "local" but rather collected the other side of the brain.
Strengths:
The authors use a cutting-edge technique.
Weaknesses:
Although the authors have toned down their claims, the statement in the title ("Synchronous Ensembles of Hippocampal CA1 Pyramidal Neurons Associated with Theta but not Ripple Oscillations During Novel Exploration") is still unsupported.
One could write the same title while voltage imaging one mouse and recording LFP from another mouse.
To properly convey the results, the title should be modified to read
"Synchronous Ensembles of Hippocampal CA1 Pyramidal Neurons Associated with Contralateral Theta but not with Contralateral Ripple Oscillations During Novel Exploration"
Without making this change, the title - and therefore the entire work - is misleading at best.
We thank the reviewer for the thoughtful and constructive suggestion regarding the title. We fully understand the concern that our original title may have overstated the specificity of the contralateral LFP recordings, potentially allowing for misinterpretation.
In our results, synchronous ensembles are associated with intracellular theta oscillations recorded from the ipsilateral hippocampus and with extracellular theta but not ripples oscillations recorded from the contralateral hippocampus. To clarify this distinction and minimize the potential for misinterpretation, we have revised the abstract accordingly.
Abstract (line18):
“… Notably, these synchronous ensembles were not associated with contralateral ripple oscillations but were instead phase-locked to theta waves recorded in the contralateral CA1 region. Moreover, the subthreshold membrane potentials of neurons exhibited coherent intracellular theta oscillations with a depolarizing peak at the moment of synchrony.”
Based on this, we propose the following revised title, which we believe more effectively communicates the central finding of our study:
“Synchronous Ensembles of Hippocampal CA1 Pyramidal Neurons During Novel Exploration”.
Compared to the reviewer’s suggested title, this version offers a clearer and more concise summary of our findings while allowing important methodological details to be fully conveyed in the abstract and main text. While the suggested title accurately reflects the source of the LFP signals, it does not mention the intracellular theta oscillations recorded from the ipsilateral hippocampus, which are a critical part of our results. Including both the intracellular and extracellular recording contexts in the title would make it overly long and potentially less accessible to readers. In contrast, the revised title succinctly captures the core phenomenon, and the updated abstract now explicitly clarifies the relationship between the synchronous ensembles and both types of oscillatory signals.
We sincerely appreciate the reviewer’s input, which helped us refine both the language and the presentation of our findings. We hope these changes address the concern and clarify the scope of our work.
Recommendations for the authors:
Reviewer #3 (Recommendations for the authors):
(1) Change the title. Although the authors have toned down their claims, the statement in the title ("Synchronous Ensembles of Hippocampal CA1 Pyramidal Neurons Associated with Theta but not Ripple Oscillations During Novel Exploration") is still unsupported. One could write the same title while voltage imaging one mouse and recording LFP from another mouse. To properly convey the results, the title should be modified to read
"Synchronous Ensembles of Hippocampal CA1 Pyramidal Neurons Associated with Contralateral Theta but not with Contralateral Ripple Oscillations During Novel Exploration"
Without making this change, the title - and therefore the entire work - is misleading at best. But if you can manage that (and attend to comment #2 below), then the manuscript would not be making any false statements.
Please see our reply in the public review above.
(2) Report the exact locations of the contralateral recording electrodes. In their rebuttal, the authors supplies a figure ("Author response image 1") in which they show damage to the neocortex and fluorescence signal in the CA1 pyramidal cell layer. This is useful, but it is unclear from which animal this histology was generated.
Please include this (or another similar) photograph in Figure 1B, right next to the voltage imaging photograph. Indicate from which animal each photograph was obtained - ideally, provide the two photographs from the same animal. Second, please include such paired photographs - along with paired signals - for every animal that you are able to.
If you can manage that, it will add credibility to the statement that the recordings are indeed from the contralateral CA1 pyramidal cell layer (as opposed to from the contralateral hemisphere).
We thank the reviewer for this important point. We have followed the suggestion and now provide paired photographs showing LFP electrode tracks and voltage images from the same animal (see revised Figure 1B)
In addition, we have included similar paired photographs for additional animals used in this study (see Figure 1-figure supplement 1).
These updates directly support the claim that LFP recordings were obtained from the contralateral CA1 pyramidal layer, rather than from the contralateral hemisphere. We sincerely thank the reviewer for the valuable suggestion, which has substantially strengthened our manuscript.
