Neurons in the medial prefrontal cortex that are not modulated by hippocampal sharp-wave ripples are involved in spatial tuning and signaling upcoming choice

Reviewer #1 (Public review):

Summary:

The authors used high-density probe recordings in the medial prefrontal cortex (PFC) and hippocampus during a rodent spatial memory task to examine functional sub-populations of PFC neurons that are modulated vs. unmodulated by hippocampal sharp-wave ripples (SWRs), an important physiological biomarker that is thought to have a role in mediating information transfer across hippocampal-cortical networks for memory processes. SWRs are associated with the reactivation of representations of previous experiences, and associated reactivation in hippocampal and cortical regions has been proposed to have a role in memory formation, retrieval, planning, and memory-guided behavior. This study focuses on awake SWRs that are prevalent during immobility periods during pauses in behavior. Previous studies have reported strong modulation of a subset of prefrontal neurons during hippocampal SWRs, with some studies reporting prefrontal reactivation during SWRs that have a role in spatial memory processes. The study seeks to extend these findings by examining the activity of SWR-modulated vs. unmodulated neurons across PFC sub-regions, and whether there is a functional distinction between these two kinds of neuronal populations with respect to representing spatial information and supporting memory-guided decision-making.

Strengths:

The major strength of the study is the use of Neuropixels 1.0 probes to monitor activity throughout the dorsal-ventral extent of the rodent medial prefrontal cortex, permitting an investigation of functional distinction in neuronal populations across PFC sub-regions. They are able to show that SWR-unmodulated neurons, in addition to having stronger spatial tuning than SWR-modulated neurons as previously reported, also show stronger directional selectivity and theta-cycle skipping properties.

Weaknesses:

(1) While the study is able to extend previous findings that SWR-modulated PFC neurons have significantly lower spatial tuning that SWR-unmodulated neurons, the evidence presented does not support the main conclusion of the paper that only the unmodulated neurons are involved in spatial tuning and signaling upcoming choice, implying that SWR-modulated neurons are not involved in predicting upcoming choice, as stated in the abstract. This conclusion makes a categorical distinction between two neuronal populations, that SWR-modulated neurons are involved and SWR-unmodulated are not involved in predicting upcoming choice, which requires evidence that clearly shows this absolute distinction. However, in the analyses showing non-local population decoding in PFC for predicting upcoming choice, the results show that SWR-unmodulated neurons have higher firing rates than SWR-modulated neurons, which is not a categorical distinction. Higher firing rates do not imply that only SWR-unmodulated neurons are contributing to the non-local decoding. They may contribute more than SWR-modulated neurons, but there are no follow-up analyses to assess the contribution of the two sub-populations to non-local decoding.

(2) Further, the results show that during non-local representations of the hippocampus of the upcoming options, SWR-excited PFC neurons were more active during hippocampal representations of the upcoming choice, and SWR-inhibited PFC neurons were less active during hippocampal representations of the alternative choice. This clearly suggests that SWR-modulated neurons are involved in signaling upcoming choice, at least during hippocampal non-local representations, which contradicts the main conclusion of the paper.

(3) Similarly, one of the analyses shows that PFC nonlocal representations show no preference for hippocampal SWRs or hippocampal theta phase. However, the examples shown for non-local representations clearly show that these decodes occur prior to the start of the trajectory, or during running on the central zone or start arm. The time period of immobility prior to the start arm running will have a higher prevalence of SWRs and that during running will have a higher prevalence of theta oscillations and theta sequences, so non-local decoded representations have to sub-divided according to these known local-field potential phenomena for this analysis, which is not followed.

(4) The primary phenomenon that the manuscript relies on is the modulation of PFC neurons by hippocampal SWRs, so it is necessary to perform the PFC population decoding analyses during SWRs (or examine non-local decoding that occurs specifically during SWRs), as reported in previous studies of PFC reactivation during SWRs, to see if there is any distinction between modulated and unmodulated neurons in this reactivation. Even in the case of independent PFC reactivation as reported by one study, this PFC reactivation was still reported to occur during hippocampal SWRs, therefore decoding during SWRs has to be examined. Similarly, the phenomenon of theta cycle skipping is related to theta sequence representations, so decoding during PFC and hippocampal theta sequences has to be examined before coming to any conclusions.

Reviewer #2 (Public review):

Summary:

This work by den Bakker and Kloosterman contributes to the vast body of research exploring the dynamics governing the communication between the hippocampus (HPC) and the medial prefrontal cortex (mPFC) during spatial learning and navigation. Previous research showed that population activity of mPFC neurons is replayed during HPC sharp-wave ripple events (SWRs), which may therefore correspond to privileged windows for the transfer of learned navigation information from the HPC, where initial learning occurs, to the mPFC, which is thought to store this information long term. Indeed, it was also previously shown that the activity of mPFC neurons contains task-related information that can inform about the location of an animal in a maze, which can predict the animals' navigational choices. Here, the authors aim to show that the mPFC neurons that are modulated by HPC activity (SWRs and theta rhythms) are distinct from those "encoding" spatial information. This result could suggest that the integration of spatial information originating from the HPC within the mPFC may require the cooperation of separate sets of neurons.

This observation may be useful to further extend our understanding of the dynamics regulating the exchange of information between the HPC and mPFC during learning. However, my understanding is that this finding is mainly based upon a negative result, which cannot be statistically proven by the failure to reject the null hypothesis. Moreover, in my reading, the rest of the paper mainly replicates phenomena that have already been described, with the original reports not correctly cited. My opinion is that the novel elements should be precisely identified and discussed, while the current phrasing in the manuscript, in most cases, leads readers to think that these results are new. Detailed comments are provided below.

Major concerns:

(1) The main claim of the manuscript is that the neurons involved in predicting upcoming choices are not the neurons modulated by the HPC. This is based upon the evidence provided in Figure 5, which is a negative result that the authors employ to claim that predictive non-local representations in the mPFC are not linked to hippocampal SWRs and theta phase. However, it is important to remember that in a statistical test, the failure to reject the null hypothesis does not prove that the null hypothesis is true. Since this claim is so central in this work, the authors should use appropriate statistics to demonstrate that the null hypothesis is true. This can be accomplished by showing that there is no effect above some size that is so small that it would make the effect meaningless (see https://doi.org/10.1177/070674370304801108).

(2) The main claim of the work is also based on Figure 3, where the authors show that SWRs-unmodulated mPFC neurons have higher spatial tuning, and higher directional selectivity scores, and a higher percentage of these neurons show theta skipping. This is used to support the claim that SWRs-unmodulated cells encode spatial information. However, it must be noted that in this kind of task, it is not possible to disentangle space and specific task variables involving separate cognitive processes from processing spatial information such as decision-making, attention, motor control, etc., which always happen at specific locations of the maze. Therefore, the results shown in Figure 3 may relate to other specific processes rather than encoding of space and it cannot be unequivocally claimed that mPFC neurons "encode spatial information". This limitation is presented by Mashoori et al (2018), an article that appears to be a major inspiration for this work. Can the authors provide a control analysis/experiment that supports their claim? Otherwise, this claim should be tempered. Also, the authors say that Jadhav et al. (2016) showed that mPFC neurons unmodulated by SWRs are less tuned to space. How do they reconcile it with their results?

(3) My reading is that the rest of the paper mainly consists of replications or incremental observations of already known phenomena with some not necessarily surprising new observations:
a) Figure 2 shows that a subset of mPFC neurons is modulated by HPC SWRs and theta (already known), that vmPFC neurons are more strongly modulated by SWRs (not surprising given anatomy), and that theta phase preference is different between vmPFC and dmPFC (not surprising given the fact that theta is a travelling wave).
b) Figure 4 shows that non-local representations in mPFC are predictive of the animal's choice. This is mostly an increment to the work of Mashoori et al (2018). My understanding is that in addition to what had already been shown by Mashoori et al here it is shown how the upcoming choice can be predicted. The author may want to emphasize this novel aspect.
c) Figure 6 shows that prospective activity in the HPC is linked to SWRs and theta oscillations. This has been described in various forms since at least the works of Johnson and Redish in 2007, Pastalkova et al 2008, and Dragoi and Tonegawa (2011 and 2013), as well as in earlier literature on splitter cells. These foundational papers on this topic are not even cited in the current manuscript.
Although some previous work is cited, the current narrative of the results section may lead the reader to think that these results are new, which I think is unfair. Previous evidence of the same phenomena should be cited all along the results and what is new and/or different from previous results should be clearly stated and discussed. Pure replications of previous works may actually just be supplementary figures. It is not fair that the titles of paragraphs and main figures correspond to notions that are well established in the literature (e.g., Figure 2, 2nd paragraph of results, etc.).
d) My opinion is that, overall, the paper gives the impression of being somewhat rushed and lacking attention to detail. Many figure panels are difficult to understand due to incomplete legends and visualizations with tiny, indistinguishable details. Moreover, some previous works are not correctly cited. I tried to make a list of everything I spotted below.

Author response:

We thank the reviewers for their thoughtful feedback. Below we provide an initial response to the central concerns that they have raised. In general, as part of our revisions, we plan to perform additional analyses to strengthen our conclusions, tone down more speculative interpretations, and clarify the novel contributions of our work. A full, point-by-point reply will follow alongside the revised manuscript.

Briefly, the reviewers’ central concerns are that some of the conclusions are not sufficiently supported by the experimental evidence, specifically (1) the involvement of sharp-wave ripple (SWR)-unmodulated PFC neurons in signaling upcoming choice and (2) the absence of SWR time-locking of PFC non-local representations. They further suggest that (3) the spatial tuning in the PFC may reflect other cognitive processes rather than encoding spatial information; and (4) the manuscript is ambiguous as to which results are novel or corroborating previous work.

(1) SWR-unmodulated PFC neurons signaling upcoming choice

Reviewer 1 suggests that our finding that SWR-modulated neurons relate to hippocampal non-local representations contradicts the manuscript’s main conclusion. However, in our view, there is no contradiction and the finding highlights the distinction between the two sub-populations, namely the SWR-modulated neurons linked to hippocampal non-local representations, and the SWR-unmodulated neurons that are more active during prefrontal non-local representations.

We do agree with the reviewer that the observation of higher firing rates of SWR-unmodulated neurons in the expression of non-local representations does not mean that these neurons are the sole or even main contributors to the non-local decoding. To address both comments, we will perform additional analyses to further disentangle the contributions of SWR-modulated and SWR-unmodulated PFC neurons to the non-local representations of upcoming choice.

(2) Time-locking of PFC non-local representations to hippocampal SWRs

Reviewer 1 comments that in the analysis of time-locking to hippocampal SWRs and theta phase, the behavior of the animals needs to be taken into account (i.e., immobility or running). We confirm that this was indeed done in our analysis and we will clarify this point in the revised manuscript.

The reviewer further requested that PFC decoding during SWRs be performed at shorter timescales as in previous studies. We like to point out that (1) we found no increase in non-local decoding in the PFC around SWR onset (see Fig 5a), and (2) most of the non-local representations in the PFC occurred during the expression of local representations in the hippocampus (see Fig 4d). These data suggest that the non-local representations in both brain regions are expressed independently. To further strengthen this idea, we plan to (1) include the result of decoding PFC activity during SWRs at fine timescales as the reviewer suggested, and (2) look at the firing rates of PFC neurons during non-local representations exclusively when the hippocampus is encoding the actual (local) position.

Following a suggestion by reviewer 2, we will also add a statistical assessment of how strongly the data supports the absence of time-locking.

(3) Spatial tuning in the mPFC

Reviewer 2 points out that the spatial tuning in the prefrontal cortex may be related to cognitive processes (e.g., attention or decision-making) rather than spatial encoding. However, our results show that decoded mPFC activity reliably differentiates between the two start and goal arms (Fig 4a), rate maps show little evidence of mirroring (Fig 3a), and the activity predicts turns in the cue-based task during which goal arms switch pseudo-randomly (meaning that the non-local representations encode the North and South arm alternatingly and correctly, rather than encoding a general rewarded goal arm; Fig. 4b). While it is likely that mPFC encodes several task-related variables, our data suggest that it also encodes distinct locations.

The reviewer further claims that the results of Jadhav et al. (2016) contradict our findings because they supposedly showed that mPFC neurons unmodulated by SWRs are less tuned to space. However, this is incorrect, as Jadhav et al. (2016) showed that SWR-unmodulated PFC neurons have lower spatial coverage and consequentially are more spatially selective, which is consistent with our observations. We will rephrase this in the text to improve clarity.

(4) Novelty

We thank reviewer 2 for pointing out the significance of several novel findings in our work that deserve to be highlighted. This includes the dorsal-ventral profile of SWR-modulation and theta phase locking in the PFC and our observation that the neural representations in the PFC precede the behavioral switch in reversal learning. In our revised manuscript, we will rewrite the text to better emphasize our novel contributions, clearly distinguish new findings from confirmatory observations, and add missing citations where appropriate.