Functional specialization of mPFC–BLA and mPFC–NAc pathways in affective state representation

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 of 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 in 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 limiting generalizability and leaving the readers with questions about the impact of sex differences on their results. The tube test is used as a manipulation of the "emotional state" in several of the experiments. While the authors show the changes to corticosterone levels as a consequence of win/loss in the tube test, stronger claims might be made with comparisons to other gold standard stressors such as forced social defeat or social isolation.

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 appear 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, this is important to understand the "neural code" within the PFC that contributes to such behaviours and how it is relayed to other brain structures.

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 BLA or NAc or the diversity of projection neuron subtypes that mediate these pathways. This is an important future direction for this work but does not detract from the main finding as reported. The electrophysiological data in Figure 7 have significant experimental confounds that makes their interpretation challenging.

Author Response:

The following is the authors’ response to the original reviews.

Public review:

Reviewer #1 (Public review):

Weaknesses:

The authors focused primarily on female mice without commenting on the effect that sex differences would have on their results.

We agree that sex is an important biological variable. Our experiments were performed primarily in female mice to align with the higher prevalence of affective disorders in females and to maintain consistency across experiments. We now explicitly acknowledge this as a limitation in the Discussion and note that future studies will be needed to determine whether the projection-specific coding principles identified here generalize to male animals. Relevant literature on sex-specific mPFC→BLA/NAc function has also been incorporated.

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.

We appreciate the reviewer’s concern regarding the use of the term “emotional states.” In the revised manuscript, we have clarified our terminology and now use “behavioral states associated with affective valence” where appropriate. We have also added references supporting the use of open field center vs. corner occupancy, elevated plus maze performance, and social interaction assays as established proxies for anxiety-like and affect-related behaviors.

Importantly, to provide physiological support for these interpretations, we now include data showing that repeated win/loss outcomes in the tube test are associated with increased corticosterone levels in loser mice. These results indicate that the behavioral manipulations used in this study are accompanied by measurable physiological changes linked to stress-related processes.

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.

We agree that detailed reporting of projection targeting is important for reproducibility. We have expanded the Methods and Results to more clearly describe viral targeting, recording locations, and histological verification of mPFC projections to the lateral BLA and NAc shell. We also now explicitly acknowledge the anatomical and cellular heterogeneity within these regions as a limitation and discuss this as an important direction for future work.

The population-level analysis in the manuscript requires more rigor to reduce bias and statistical controls for establishing the significance of their results.

We have strengthened the statistical analyses throughout the manuscript. Specifically, we have incorporated permutation-based controls for key analyses, clarified how behavioral and neural features were defined, and provided additional details on dimensionality reduction and clustering approaches. Exact p values, sample sizes, and statistical tests are now reported throughout the manuscript and figure legends.

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.

We agree that the tube test is not a classical chronic stress paradigm such as social defeat. In our study, the tube test was used to establish social hierarchy rather than to model sustained stress. We have revised the manuscript to clarify this point and have tempered our language accordingly. At the same time, our corticosterone measurements indicate that repeated social competition induces measurable physiological changes, suggesting that the paradigm captures aspects of social hierarchy–related stress. We now frame these effects conservatively and acknowledge the need for future studies using additional stress paradigms.

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.

We have revised all figures to include individual data points (scatter overlays) wherever appropriate and have improved reporting of statistical details, including exact p values and group sizes, to enhance transparency and reproducibility.

Taken together, these revisions clarify our interpretations, improve methodological transparency, and strengthen the rigor of the analyses while preserving the main conclusions of the study.

Reviewer #2 (Public Review):

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.

We agree that both the basolateral amygdala (BLA) and nucleus accumbens (NAc) are highly heterogeneous. Our study was designed to focus on projection-defined mPFC outputs (presynaptic activity) rather than resolving postsynaptic subregional or cell-type diversity. We have now:

- Clarified targeting strategies (PL→NAc shell and PL→BLA basal region)

- Added histological descriptions of injection and recording sites

- Expanded the Discussion to acknowledge how subregional and cellular heterogeneity may contribute to the observed variability

We also highlight this as an important direction for future work.

The electrophysiological data have significant experimental confounds and more methodological information is required to support other conclusions related to these data.

We have significantly strengthened the electrophysiological component by:

- Providing detailed recording conditions (access resistance, membrane properties, inclusion criteria)

- Clarifying stimulus protocols and normalization procedures

- Including representative traces and quantification of exclusion rates

- Addressing potential confounds such as viral expression variability and stimulation parameters

These revisions improve both interpretability and reproducibility of the electrophysiological findings.

Reviewer #3 (Public Review):

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.

We agree that sex is an important biological variable. We have now clearly specified throughout the manuscript that experiments were performed primarily in female mice and have added a rationale for this choice in the Methods. Briefly, we focused on females to align with the higher prevalence of affective disorders in females and to maintain consistency across experiments. We now explicitly acknowledge this as a limitation in the Discussion and note that future studies will be needed to determine whether these findings generalize to male animals.

(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.

We thank the reviewer for raising this important point. Mice lacking identifiable center-ON neurons were excluded from analyses that specifically relied on this functional classification, as inclusion of such datasets would preclude meaningful comparison of this neuronal population. We have now clarified this criterion in the Methods and Results. Importantly, this exclusion does not affect analyses performed at the population level or those not dependent on center-ON classification. We now explicitly discuss this limitation and note that variability in the presence of center-ON neurons may reflect biological heterogeneity across animals.

(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.

We have added baseline comparisons of mPFC→BLA and mPFC→NAc activity across subjects to control for inter-individual variability and better contextualize behavior-related changes.

(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.

We now provide detailed descriptions of experimental design, including habituation, randomized testing order, and recovery periods between assays. We also discuss potential cumulative stress effects as a limitation.

(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.

We thank the reviewer for this important clarification. We agree that grooming can be associated with both stress-related and self-soothing behaviors. In the revised manuscript, we have clarified that grooming is not strictly a “non-anxiety” behavior but instead represents a distinct behavioral state that may reflect stress regulation or internal state transitions. We have revised the text accordingly to avoid oversimplification and to better align with the literature.

(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.

We agree that stronger integration between neural and behavioral findings would strengthen the manuscript. In the revised version, we have added quantitative analyses examining the similarity and divergence of activity patterns across behavioral contexts (e.g., cross-context comparisons and correlation-based analyses). We have also expanded the Discussion to better integrate our findings with prior studies on mPFC→NAc and mPFC→BLA pathways in reward, aversion, and social behavior, thereby providing a more cohesive interpretation of pathway-specific functions.

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.

We have addressed all these minor concerns, including:

- Clarifying whether the same mice were used across assays

- Reporting sample sizes for each experiment

- Improving figure clarity (e.g., scaling, labeling, scatter points)

- Providing details for social stimuli (M1 vs. M2)

- Expanding statistical methods and animal selection criteria

Summary

In summary, we have made substantial revisions to:

- Improve conceptual precision (behavior vs. emotional state)

- Increase methodological transparency and statistical rigor

- Strengthen physiological validation

- Clarify experimental design and limitations

- Enhance integration with existing literature

We believe these revisions significantly improve the clarity, rigor, and interpretability of the manuscript, and we are grateful for the reviewers’ guidance in strengthening this work.