Decoding the hidden variabilities in mPFC descending pathways across emotional states

Reviewer #1 (Public review):

Summary:

It is well known that neurons in the medial prefrontal cortex (mPFC) are involved in higher cognitive functions such as executive planning, motivational processing, and internal state-mediated decision-making. These internal states often correlate with the emotional states of the brain. While several studies point to the role of mPFC in regulating behavior based on such emotional states, the diversity of information processing in its sub-populations remains a less explored territory. In this study, the authors try to address this gap by identifying and characterizing some of these sub-populations in mice using a combination of projection-specific imaging, function-based tagging of neurons, multiple behavioral assays, and ex-vivo patch clamp recordings.

Strengths:

The authors targeted mPFC projections to the nucleus accumbens (NAc) and basolateral amygdala (BLA). Using the open field task (OFT), the authors identified four relevant behavioral states as well as neurons active while the animal was in the center region ("center-ON neurons"). By characterizing single-unit activity and using dimensionality reduction, the authors show differentiated coding of behavioral events at both the projection and functional levels. They further substantiate this effect by showing higher sensitivity of mPFC-BLA center-ON neurons during time spent in the open arms of the elevated plus maze (EPM). The authors then pivoted to the three-chamber social interaction (SI) assay to show the different subsets of neurons encode preference for social stimulus over non-social. This reveals an interesting diversity in the function of these sub-populations on multiple levels. Lastly, the authors used the tube test as a manipulation of the anxiety state of mice and compared behavioral differences before/after the OFT and social interaction tasks. This experiment revealed that "losers" of the tube test spend less time in the center of the open field while "winners" show a stronger preference for the familiar mouse over the object. Using patch-clamp experiments, the authors also found that "winners" exhibit stronger synaptic transmission in the mPFC-NAc projection while "losers" exhibit stronger synaptic transmission in the mPFC-BLA projection. Given the popularity of the tube test assay in rank determination, this provides useful insights into possible effects on anxiety levels and synaptic plasticity. Overall, the many experiments performed by the authors reveal interesting differences in mPFC neurons relative to their involvement in high or low anxiety behaviors, social preference, and social rank.

Weaknesses:

The authors focused primarily on female mice without commenting on the effect that sex differences would have on their results. While the authors have identified relevant behavioral states across the various behavioral tasks, there is still a missing link between them and "emotional states" - the phrase used by them emphatically throughout the manuscript. The authors have neither provided adequate references to satisfy this gap nor shared any data pertaining to relevant readouts such as cortisol levels. Both the projection-specific recordings and patch-clamp experiments, including histology reports in the manuscript, would provide essential information for anyone trying to replicate the results, especially since it's known that sub-populations in the BLA and NAc can have vastly different functions. The population-level analysis in the manuscript requires more rigor to reduce bias and statistical controls for establishing the significance of their results. Lastly, the tube test is used as a manipulation of the "emotional state" in several of the experiments. While the tube test can cause a temporary spike in anxiety of the participating mice, it is not known to produce a sustained effect - unless there are additional interventions such as forced social defeat. Thus, additional controls for these experiments are essential to support claims based on changes in the emotional state of mice. Apart from the methodology, the manuscript could also be improved with the addition of clear scatter points in all the plots along with detailed measures of the statistical tests such as exact p values and size of groups being compared.

Reviewer #2 (Public review):

Summary:

The goal of this proposal was to understand how two separate projection neurons from the medial prefrontal cortex, those innervating the basolateral amygdala (BLA ) and nucleus accumbens (NAc), contribute to the encoding of emotional behaviors. The authors record the activity of these different neuron classes across three different behavioral environments. They propose that, although both populations are involved in emotional behavior, the two populations have diverging activity patterns in certain contexts. A subset of projections to the NAc appears particularly important for social behavior. They then attempt to link these changes to the emotional state of the animal and changes in synaptic connectivity.

Strengths:

The behavioral data builds on previous studies of these projection neurons supporting distinct roles in behavior and extend upon previous work by looking at the heterogeneity within different projection neurons across contexts.

Weaknesses:

The diversity of neurons mediating these projections and their targeting within the BLA and NAc is not explored. These are not homogeneous structures and so one possibility is that some of the diversity within their findings may relate to targeting of different sub-structures within each region. The electrophysiological data have significant experimental confounds and more methodological information is required to support other conclusions related to these data.

Reviewer #3 (Public review):

Summary:

This manuscript investigates the distinct contributions of mPFC→BLA and mPFC→NAc pathways in emotional regulation, with implications for understanding anxiety, exploration, and social preference behaviors. Using Ca2+ imaging, optogenetics, and patch-clamp recording, the authors demonstrate pathway-specific roles in encoding emotional states of opposite valence. They further identify subsets of neurons ("center-ON") with heightened activity under anxiety-inducing conditions. These findings challenge the traditional view of functional similarity between these pathways and provide valuable insights into neural circuit dynamics relevant to emotional disorders.

The study is well-designed and addresses an important topic, but several methodological and interpretational issues require clarification to strengthen the conclusions.

Weaknesses:

Major Weaknesses:

(1) The manuscript does not clearly and consistently specify the sex of the mice used for behavioral and imaging experiments. Given the known influence of sex on emotional behaviors and neural activity, this omission raises concerns about the generalizability of the findings. The authors should make clear throughout the manuscript whether male, female, or mixed-sex cohorts were used and provide a rationale for their choice. If only one sex was used, the potential limitations of this approach should be explicitly discussed.

(2) Mice lacking "center-ON" neurons were excluded from analysis, yet the manuscript draws broad conclusions about the encoding of emotional states by mPFC pathways. It is critical to justify this exclusion and discuss how it may limit the generalizability of the findings. The inclusion of data or contextualization for animals without center-ON neurons would strengthen the interpretation.

(3) The manuscript lacks baseline activity comparisons for mPFC→BLA and mPFC→NAc pathways across subjects. Providing baseline data would contextualize the observed activity changes during behavior testing and help rule out inter-individual variability as a confounding factor.

(4) Extensive behavioral testing across multiple paradigms may introduce stress and fatigue in the animals, which could confound the induction of emotional states. The authors should describe the measures taken to minimize these effects (e.g., recovery periods, randomized testing order) and discuss their potential impact on the results.

(5) Grooming is described as a "non-anxiety" behavior, which conflicts with its established role as a stress-relieving behavior that may indicate anxiety. This discrepancy requires clarification, as the distinction is central to the conclusions about the mPFC→BLA pathway's role in differentiating anxiety-related and non-anxiety behaviors.

(6) While the study highlights pathway-specific neural activity, it lacks a cohesive integration of these findings with the behavioral data. Quantifying the overlap or decorrelation of neuronal activity patterns across tasks would solidify claims about the specialization of mPFC→NAc and mPFC→BLA pathways. Likewise, the discussion should be expanded to place these findings in light of prior studies that have probed the roles of these pathways in social/emotion/valence-related behaviors.

Minor Weaknesses:

(1) The manuscript does not explicitly state whether the same mice were used across all behavioral assays. This information is critical for evaluating the validity of group comparisons. Additionally, more detail on sample sizes per assay would improve the manuscript's transparency.

(2) In Figure 2G, the difference between BLA and NAc activity during exploratory behaviors (sniffing) is difficult to discern. Adjusting the scale or reformatting the figure would better illustrate the findings.

(3) While the characteristics of the first social stimulus (M1) are specified, there is no information about the second social stimulus (M2). This omission makes it difficult to fully interpret the findings from the three-chamber test.

(4) The methods section lacks detailed information about statistical approaches and animal selection criteria. Explicitly outlining these procedures would improve reproducibility and clarity.

Author response:

Reviewing editor comments:

Overall, the reviewers found the imaging data to be strong but identified the physiology experiments as the weakest aspect of the study. Please consider either removing Figures 7 and 8 from the manuscript or significantly revising the data. If you choose to revise these figures, refer to the specific reviewer comments addressing them. Additionally, several reviewers noted that the prior literature was not adequately cited, so please consider addressing this concern.

As noted below, we will work to strengthen the physiological side of the study and ensure that we are more scrupulous in citing the prior literature. Below we summarize the major concerns of each reviewer and outline our proposed response.

Reviewer #1:

(1) Sex differences and generalizability

Various studies have shown sex differences in emotional responses and neural activity in mice, but to study both male and female mice would have required much larger numbers of mice than we could accommodate for practical reasons, so we chose to use only female mice to lay a solid foundation for future studies that compare (and perhaps contrast) males.

We will:

Make clear in the main text that we used only females.

Cite literature on sex-specific mPFC-BLA/NAc functions in the Discussion.

(2) Missing link between behavioral states and "emotional states"...relevant readouts such as cortisol

We appreciate the reviewer pointing out this inadvertent conceptual slippage. We will:

Include corticosterone measurements using an ELISA kit from archived plasma samples (collected before and after OFT/EPM tests) to correlate with behavioral and neural activity (approach refers to Panczyszyn-Trzewik et al., Steroids, 2024).

Be more precise in our language to differentiate behavioral correlates from inferred emotional states.

Carefully review the literature on OFT center time, EPM open-arm exploration, and tube test outcomes as anxiety/social hierarchy indicators and decide the best interpretation for our findings.

(3) Improve methodological detail and rigor of population-level analysis

We will:

Expand the methods section with electrophysiology parameters (e.g., access resistance criteria, stimulus protocols).

Add detailed histology figures (viral targeting, electrode placements) for mPFC-BLA/NAc projections.

Include raw data points in all plots and report exact p-values, effect sizes, and group sizes (e.g., n = 12 cells from 4 mice).

To enhance statistical rigor, we will provide clearer scatter plots with individual data points, report exact p-values, and specify group sizes in all figures.

(4) Acute vs. sustained effects after tube test and additional controls

We would like to clarify that we used repeated tube tests (3 times a day and continuing for 7 days) for assessing sustained rank effects. To address concerns about sustained emotional state changes post-tube test, we will:

Assess corticosterone levels pre/post-tube test (approach refers to Panczyszyn-Trzewik et al., Steroids, 2024).

Discuss the transient nature of hierarchy effects and cite studies using repeated tube tests for sustained rank effects.

Reviewer #2:

(1) Sub-region targeting in BLA/NAc

Although different subregions within the BLA and NAc receive distinct inputs and exhibit diverse functions, comparing neuronal activity across these subregions is beyond the scope of this paper. Our primary focus is on mPFC projections, emphasizing presynaptic activity rather than postsynaptic activity within the NAc and BLA. We focused on the PL-NAc shell and PL-BLA (BA) regions because PL-to-NAc shell projections in mice are well-documented, particularly in studies utilizing viral tracers and optogenetic tools (Britt et al., Neuron, 2012; Bossert et al., J. Neurosci., 2012). These projections regulate aversive behaviors, stress responses, and motivational states and are implicated in drug-seeking behaviors and emotional valence encoding (Jocelyn & Berridge, Biol. Psychiatry, 2013; Fetcho et al., Nat. Commun., 2023; Capuzzo & Floresco, J. Neurosci., 2020; Xie et al., BioRxiv., 2025; Domingues et al., Nat Commun., 2025). The PL-BLA projection in turn sends topographically organized projections to BLA subregions, primarily targeting the basal (BA) nuclei of the BLA (McGarry & Carter, J. Neurosci., 2016; Hoover & Vertes, Brain Struct. Funct., 2007). Both the recorded NAc shell and BLA subregions are involved in emotional valence encoding.

A detailed comparison of neuronal activity across different NAc shell and BLA subregions or comparing different cell types, such as NAc shell D1- and D2-medium spiny neurons, could each be the subject of a whole other study. Nevertheless,

We will discuss how sub-region connectivity could contribute to observed heterogeneity in the discussion, citing relevant studies, and make sure we clarify our rationale for our experimental design.

(2) Electrophysiological confounds

To strengthen the rationale for our patch-clamp recordings, we will:

Clarify in methods that recordings were performed in acute slices from behaviorally naive mice (post-tube test) to isolate synaptic changes.

Include access resistance and cell health criteria (e.g., resting membrane potential, input resistance ranges), along with precise optogenetic stimulus protocols.

Add example traces of mEPSCs/mIPSCs and quantify exclusion rates.

Reviewer #3:

(1) Specify the sexes used throughout the manuscript.

We will make this clear throughout the paper.

(2) Exclusion of mice lacking "center-ON" neurons

We will:

Explain the exclusion of mice that lacked center-ON neurons. We will also discuss the potential interpretations (e.g., floor effects in anxiety tasks) in the limitations section.

(3) Baseline activity comparisons

We will:

Add baseline neuronal activity comparison between mPFC-BLA and mPFC-NAc neurons.

(4) Stress from repeated behavioral testing

We will:

Clarify our experimental design to state how we tried to minimize the stress caused by multiple behavioral assays.

Include pre-test habituation protocols in methods.

Discuss potential cumulative stress effects in limitations.

(5) Grooming classification

While the reviewer is correct that grooming can be a stress-relieving behavior, it also obviously has many other functions, from the pragmatic to the social. In our study grooming occurred primarily in the periphery of the open field test, where it was exhibited as a behavior corresponding to neural activity patterns that differed from that which occurred in the center. As we classify the behavior in the center zone of the open field test as anxiety-like, we interpreted the peripheral grooming as indicative of the animal's adjustment to a novel environment, as suggested by previous work (Estanislau et al., Neurosci. Res., 2013; Rojas-Carvajal et al., Animal Behaviour, 2018). The nature of the grooming was primarily rostral body-licking, which accords with what Rojas-Carvajal et al. calls a “de-arousal inhibition system” that subserves novelty habituation. The duration and nature of this behavior are, interestingly enough, influenced by whether the mouse or rat lived in an enriched environment prior to the OFT (enriched environments made them quicker to explore a new environment but also quicker to get bored - no surprise, really).

We did not explain any of this in the manuscript, however, so in our revision, we will make sure to discuss these nuances and cite the relevant literature.

(6) Integrate neuronal activity and behavioral data

We will:

Include additional analyses quantifying neuronal activity overlap across tasks and refine our Discussion to better integrate these findings with prior literature.

Perform cross-correlation analyses to quantify activity overlap between OFT, EPM, and SI tasks.

Minor weaknesses

- Clarify the cohorts of mice that were used for each behavioral assay.

- Adjust Figure 2G scale and add insets to highlight sniffing differences.

- Specify that M1/M2 were age-/sex-matched unfamiliar mice in the three-chamber test.

- Detail statistical tests (e.g., mixed-effects models) and animal selection criteria in methods.

We believe these revisions will address the reviewers’ major concerns and significantly improve the manuscript. We welcome further feedback on these plans and will provide updated figures/data for the resubmission.