Synchronous Ensembles of Hippocampal CA1 Pyramidal Neurons Associated with Theta but not Ripple Oscillations During Novel Exploration

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-of-the-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.

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.

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.

Author response:

The following is the authors’ response to the previous 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.

We thank the reviewer for a thorough review of our manuscript 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 allow 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.

We thank the reviewer for a thorough review of our manuscript and for recognizing the strength of our study.

Weaknesses:

The evidence provided is weak, with the authors making surprising population-level claims based on a very sparse data set (5 data sets, each with less than 20 neurons simultaneously recorded) acquired with exciting, but less tested technology. Further, while the authors link these observations to the novelty of the context, both in the title and text, they do not include data from subsequent visits to support this. Detailed comments are below:

(1) My first question for the authors, which is not addressed in the discussion, is why these events have not been observed in the countless extracellular recording experiments conducted in rodent CA1 during exploration of novel environments. Those data sets often have 10x the neurons simultaneously recording compared to these present data, thus the highly synchronous firing should be very hard to miss. Ideally, the authors could confirm their claims via the analysis of publicly available electrophysiology data sets. Further, the claim of high extra-SWR synchrony is complicated by the observation that their recorded neurons fail to spike during the limited number of SWRs recorded during behavior- again, not agreeing with much of the previous electrophysiological recordings.

(2) The authors posit that these events are linked to the novelty of the context, both in the text, as well as in the title and abstract. However they do not include any imaging data from subsequent days to demonstrate the failure to see this synchrony in a familiar environment. If these data are available it would strengthen the proposed link to novelty is they were included.

(3) In the discussion the authors begin by speculating the theta present during these synchronous events may be slower type II or attentional theta. This can be supported by demonstrating a frequency shift in the theta recording during these events/immobility versus the theta recording during movement. (4) The authors mention in the discussion that they image deep layer PCs in CA1, however this is not mentioned in the text or methods. They should include data, such as imaging of a slice of a brain post-recording with immunohistochemistry for a layer specific gene to support this.

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 our efforts in revising the manuscript.

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 potentials (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.

We thank the reviewer for a thoughtful review of our manuscript and for pointing out the technical strength of our study.

Weaknesses:

The two main messages of the manuscript indicated in the title are not supported by the data. The title gives two messages that relate to CA1 pyramidal neurons in behaving head-fixed mice: (1) synchronous ensembles are associated with theta (2) synchronous ensembles are not associated with ripples. The main problem with the work is that the theta and ripple signals were recorded using electrophysiology from the opposite hemisphere to the one in which the spiking was monitored. However, both rhythms exhibit profound differences as a function of location.

Theta phase changes with the precise location along the proximo-distal and dorso-ventral axes, and importantly, even reverses with depth. Because the LFP was recorded using a single-contact tungsten electrode, there is no way to know whether the electrode was exactly in the CA1 pyramidal cell layer, or in the CA1 oriens, CA1 radiatum, or perhaps even CA3 - which exhibits ripples and theta which are weakly correlated and in anti-phase with the CA1 rhythms, respectively. Thus, there is no way to know whether the theta phase used in the analysis is the phase of the local CA1 theta.

Although the occurrence of CA1 ripples is often correlated across parts of the hippocampus, ripples are inherently a locally-generated rhythm. Independent ripples occur within a fraction of a millimeter within the same hemisphere. Ripples are also very sensitive to the precise depth - 100 micrometers up or down, and only a positive deflection/sharp wave is evident. Thus, even if the LFP was recorded from the center of the CA1 pyramidal layer in the contralateral hemisphere, it would not suffice for the claim made in the title.

We thank the reviewer for pointing out the issue regarding the claim made in the title. We have revised the manuscript to clarify that the theta and ripple oscillations referenced in the title refer to specific frequency bands of intracellular and contralaterally recorded field potentials rather than field potentials recorded at the same site as the neuronal activity.

Abstract (line19):

“… 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.”

Introduction (line68):

“… Surprisingly, these synchronous ensembles occurred outside of contralateral ripples and were phase-locked to intracellular theta oscillations as well as extracellular theta oscillations recorded from the contralateral CA1 region.”

To address concerns about electrode placement, we have now included posthoc histological verification of electrode locations, confirming that they were positioned in the contralateral CA1 pyramidal layer (Author response image 1).

Author response image 1.

Post-hoc histological section showing the location of a DiI-coated electrode in the contralateral CA1 pyramidal layer. Scale bar: 300 μm.

While we appreciate that theta and ripple oscillations exhibit regional variations in phase and amplitude, previous studies have demonstrated a strong co-occurrence and synchrony of these oscillations between both hippocampi1-3. Given that our primary objective was to examine how neuronal ensembles relate to large-scale hippocampal oscillation states rather than local microcircuit-level fluctuations, we recorded theta and ripple oscillations from the contralateral CA1 region.

However, we acknowledge that contralateral recordings do not capture all ipsilateral-specific dynamics. Theta phases vary with depth and precise location, and local ripple events may be independently generated across small spatial scales. To reflect this, we have now explicitly acknowledged these considerations in the discussion.

Discussion (line527):

While contralateral LFP recordings reliably capture large-scale hippocampal theta and ripple oscillations, they may not fully account for ipsilateral-specific dynamics, such as variations in theta phase alignment or locally generated ripple events. Although contralateral recordings serve as a well-established proxy for large-scale hippocampal oscillatory states, incorporating simultaneous ipsilateral field potential recordings in future studies could refine our understanding of local-global network interactions. Despite these considerations, our findings provide robust evidence for the existence of synchronous neuronal ensembles and their role in coordinating newly formed place cells. These results advance our understanding of how synchronous neuronal ensembles contribute to spatial memory acquisition and hippocampal network coordination.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

The authors have provided sufficient experimental and analytical data addressing my comments, particularly regarding consistency with past electrophysiological data and the exclusion of potential imaging artifacts.

We thank the reviewer for recognizing our efforts in revising the manuscript.

Minor comment: In Figure 2C and Figure 5-figure supplement 1, 'paired Student's t-test' is not entirely appropriate. More precisely, either 'paired t-test' or 'Student's t-test' would better indicate the correct statistical method. Please verify whether these data comparisons are within-group or between-group.

Thank you for the comment. We have revised the manuscript as suggested.

Reviewer #2 (Recommendations for the authors):

I have no further major requests and thank the authors for the additional data and analyses.

We thank the reviewer for recognizing our efforts in revising the manuscript.

Minor points- line 169- typo, correct grant to grand

Thank you for pointing it out. The typo has been corrected.

(1) Buzsaki, G. et al. Hippocampal network patterns of activity in the mouse. Neuroscience 116, 201-211 (2003). https://doi.org:10.1016/s03064522(02)00669-3

(2) Szabo, G. G. et al. Ripple-selective GABAergic projection cells in the hippocampus. Neuron 110, 1959-1977 e1959 (2022). https://doi.org:10.1016/j.neuron.2022.04.002

(3) Huang, Y. C. et al. Dynamic assemblies of parvalbumin interneurons in brain oscillations. Neuron 112, 2600-2613 e2605 (2024). https://doi.org:10.1016/j.neuron.2024.05.015