Functional specialization of mPFC-BLA and mPFC-NAc pathways in affective state representation
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eLife Assessment
Du et al. present a valuable study examining neural activation in medial prefrontal cortex (mPFC) subpopulations projecting to the basolateral amygdala (BLA) and nucleus accumbens (NAc) during behavioral tasks assessing anxiety, social preference, and social dominance. The strength of the evidence linking in vivo neural physiology to behavioral outcomes was considered solid; however, the slice electrophysiology data and their interpretation were less well received. Overall, the reviewers felt that the revised work provides insight into how distinct mPFC→BLA and mPFC→NAc pathways influence anxiety, exploration, and social behaviors.
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Abstract
Effective emotional processing, crucial for adaptive behavior, is mediated by the medial prefrontal cortex (mPFC) via connections to the basolateral amygdala (BLA) and nucleus accumbens (NAc), traditionally considered functionally similar in modulating reward and aversion responses. However, the functional specialization of the mPFC→BLA and mPFC→NAc pathways in representing affective states remains unclear. We found that while overall firing patterns appeared consistent across emotional states, deeper analysis revealed distinct variabilities. Specifically, mPFC→BLA neurons, especially “center-ON” neurons, exhibited heightened activity during behaviors classically associated with anxiety-like states, suggesting their involvement in aversive behavioral regulation. Conversely, mPFC→NAc neurons were more active during exploratory and approach-related behaviors, implicating them in the processing of positively valenced behavioral states. Notably, mPFC→NAc neurons showed significant pattern decorrelation during social interactions, suggesting a pivotal role in processing social preference. Additionally, chronic emotional states affected these pathways differently: positively valenced contexts enhanced mPFC→NAc activity, while negatively valenced conditions boosted mPFC→BLA activity. Consistent with this behavioral divergence, repeated win/loss outcomes in the tube test were associated with elevated corticosterone levels in loser mice, indicating that repeated social competition produces measurable physiological responses linked to social hierarchy. These findings challenge the assumed functional similarity and highlight distinct correlational patterns, suggesting potential, but not yet causally established, roles of mPFC→BLA and mPFC→NAc pathways in shaping emotional states.
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eLife Assessment
Du et al. present a valuable study examining neural activation in medial prefrontal cortex (mPFC) subpopulations projecting to the basolateral amygdala (BLA) and nucleus accumbens (NAc) during behavioral tasks assessing anxiety, social preference, and social dominance. The strength of the evidence linking in vivo neural physiology to behavioral outcomes was considered solid; however, the slice electrophysiology data and their interpretation were less well received. Overall, the reviewers felt that the revised work provides insight into how distinct mPFC→BLA and mPFC→NAc pathways influence anxiety, exploration, and social behaviors.
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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 …
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 have addressed all comments.
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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 …
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 some experimental confounds that makes their interpretation challenging.
Comments on revisions:
The authors have improved the manuscript somewhat by refining their description of the results. However, the normalized EPSC experiments still do not make much sense. If you have a higher light intensity or LED duration the curve of the EPSC response will saturate earlier. Similarly, if you are in a highly, or poorly labeled slice or subregion of a slice then you will see responses emerge at different intensities based on the number of synapses labelled. There is no standardization in the way these experiments were performed, so performing some arbitrary post hoc normalisation does not correct for this. Similarly, they also place the fibreoptic manually above the slice each time. This makes it much harder to determine the actual light intensity delivered to the slice on a cell by cell and group by group basis.
I have reduced my public statement from significant experimental confounds, to some experimental confounds. But the way the experiments were performed does not allow the normalized data to really be interpretable. They still argue that normalized EPSCs are relatively larger. I don't even really understand what this means biologically.
The subsequent rise/decay and other measures is now better described. However, they note that the decay constant is larger. This means that the kinetics are slower, not enhanced, as they describe.
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Author response:
The following is the authors’ response to the previous reviews
We sincerely thank the editors and reviewers for their careful evaluation and constructive feedback, which has helped us substantially improve the clarity and rigor of the manuscript. In the revised version, we have clarified the interpretation of the electrophysiological experiments, corrected the labeling of recorded signals as light evoked EPSCs, and removed statements implying differences in absolute synaptic strength. To address concerns about the interpretation of Fig. 7, we have added quantitative analyses of EPSC kinetics and revised the text to focus on synaptic response dynamics rather than amplitude differences. We have also removed analyses that could cause confusion and expanded the Methods section to provide additional experimental details, …
Author response:
The following is the authors’ response to the previous reviews
We sincerely thank the editors and reviewers for their careful evaluation and constructive feedback, which has helped us substantially improve the clarity and rigor of the manuscript. In the revised version, we have clarified the interpretation of the electrophysiological experiments, corrected the labeling of recorded signals as light evoked EPSCs, and removed statements implying differences in absolute synaptic strength. To address concerns about the interpretation of Fig. 7, we have added quantitative analyses of EPSC kinetics and revised the text to focus on synaptic response dynamics rather than amplitude differences. We have also removed analyses that could cause confusion and expanded the Methods section to provide additional experimental details, including the optogenetic stimulation configuration in slice recordings. Together, these revisions strengthen the interpretation of the electrophysiological results and improve the overall clarity and transparency of the study.
Public Reviews:
Reviewer #1 (Public review):
Weakness:
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.
We thank the reviewer for these thoughtful comments.
First, we acknowledge that the present study was conducted primarily in female mice, which may limit the generalizability of the findings. Female mice were selected to reduce variability associated with male aggression and housing-related stress, which can complicate behavioral assays such as social interaction and dominance testing. While focusing on a single sex allowed us to maintain experimental consistency across multiple behavioral paradigms, we agree that sex differences could influence the neural circuits underlying emotional and social behaviors. We have now added a statement in the Discussion acknowledging this limitation and noting that future studies will be necessary to determine whether similar circuit mechanisms operate in male mice.
Second, we appreciate the reviewer’s suggestion regarding the use of other stress paradigms. In this study, the tube test was used primarily to establish social dominance relationships between paired mice rather than as a classical stress-induction paradigm. Nevertheless, we observed measurable physiological changes associated with repeated win/loss outcomes, including alterations in corticosterone levels in brain lysates of loser mice after repeated tube-test competitions. Notably, repeated win/loss outcomes in the tube test were associated with significant increases in corticosterone levels in loser mice, indicating that the paradigm produced measurable physiological responses consistent with stress-related processes. These findings suggest that repeated social competition in this context can induce transient physiological and behavioral changes associated with social hierarchy. We agree that paradigms such as chronic social defeat stress or social isolation represent well-established models for inducing sustained stress responses. We have therefore revised the manuscript to clarify that the tube test in our study serves as a model of social competition and rank establishment rather than a canonical stress paradigm, and we highlight the comparison with other stress models as an important direction for future work.
Recommendations for the authors:
Reviewer #2 (Recommendations for the authors):
In relation to figure 7. Their response does not really clarify the issue:
(a) They argue that they are not making claims about synapse strength. However they still state "In the mPFC→NAc pathway, blue light stimulation evoked larger excitatory postsynaptic currents (EPSCs) in winner mice compared to losers (Fig. 7E). This suggests stronger synaptic transmission in winners' mPFC→NAc circuits. " They don't show this, they just show that normalized to some arbitrary value the responses of the earlier durations is higher or lower, which is very hard to interpret.
They argue in the rebuttal that the aim of this is to highlight response kinetics, but these are not quantified or discussed in any way.
We thank the reviewer for this helpful comment. We agree that the normalized input output curves shown in the original submission did not allow conclusions about absolute synaptic strength, and we also acknowledge that response kinetics were not previously quantified despite being mentioned in the rebuttal.
To address both concerns, we have revised Fig. 7 and added quantitative analyses of EPSC kinetics. Specifically, we measured the rise and decay slopes of light-evoked EPSCs recorded in postsynaptic neurons within the NAc and BLA of winner and loser mice. In the mPFC→BLA pathway, both the EPSC rise and decay slopes were significantly increased in loser mice compared with winners (rise slope: p = 0.0138; decay slope: p = 0.0392), suggesting enhanced synaptic responsiveness and faster charge transfer kinetics in BLA neurons of losers. In contrast, in the mPFC→NAc pathway, both mEPSC rise and decay slopes were not significantly different between groups.
These results provide a quantitative characterization of synaptic response dynamics and reveal pathway-specific differences in synaptic properties associated with social hierarchy. Importantly, this analysis does not rely on amplitude normalization and therefore allows a more interpretable comparison of synaptic response profiles between groups. We have updated Fig. 7 and the corresponding Results section to include these analyses.
(b) They still haven't labeled the responses correctly. The responses in figure 7 are not "voltage spikes" but light-evoked EPSCs.
We apologize for the incorrect terminology. All instances of “voltage spikes” have been corrected to “light-evoked EPSCs” in the figure legends and text.
(c) They argue that responses do not vary across experiments/slices because they use a constant viral injection volume targeted to the same co-ordinates and identical placement of the fiber and recording location. While I am sure they aim to do that, it is almost impossible to ensure that this was identical across experiments and that the degree of opsin labelling in their slices was the same (See for example Mao et al., 2011 PMID: 21982373 who pioneer the approach of using within slice comparisons to account for this). If I understand their explanation of their strategy correctly, the authors own rebuttal highlights this point, they seem to have needed to vary the LED duration by an order of magnitude (1-10ms) to ensure reliable responses across experiments, even for the same projection.
We thank the reviewer for raising this important point. We agree that it is not possible to ensure identical opsin expression or light delivery across experiments. We have revised the manuscript to explicitly acknowledge this limitation and clarify that normalization was used to mitigate, but not eliminate, inter-slice variability. We now avoid any interpretation that relies on absolute response amplitude across animals.
Regarding “LED duration variability (1-10 ms)”, we agree that the need to adjust stimulation duration reflects variability in effective opsin activation across slices. We now clarify this point in the Methods and Results and emphasize that stimulation parameters were optimized to reliably evoke responses rather than to equate absolute light input across experiments.
Importantly, our main conclusions do not rely on absolute EPSC amplitude comparisons. Instead, they are supported by analyses that are less sensitive to variability in opsin expression or light delivery, including EPSC kinetics (rise and decay slopes), paired-pulse ratio measurements, and AMPA/NMDA ratios. These complementary measures provide a more robust characterization of synaptic properties across conditions.
(d) Similarly in Fig S6 it is unclear what they are showing. The Y axis is still labeled in pA, yet they claim this is an action potential? Also this analysis is rather irrelevant to the data shown in figure 7 as the pathway between PFC and BLA/NAc is not preserved.
We thank the reviewer for pointing out the lack of clarity in Fig. S6. We agree that it does not directly inform the interpretation of Fig. 7 and may cause confusion. To improve the clarity and focus of the manuscript, we have therefore removed Fig. S6 from the revised manuscript. The removal of this supplementary figure does not affect the main conclusions of the study.
(e) It now also seems that these experiments were performed by placing a fiber optic into the slice to elicit responses. This should be detailed in the methods.
We thank the reviewer for noting this omission. We have added a detailed description of fiber-optic placement within the slice for optogenetic stimulation to the Methods section. Specifically, we clarify that blue light was delivered through a fiber optic positioned above the recorded slice to activate ChR2-expressing mPFC axon terminals within the BLA or NAc. The placement of the fiber relative to the recorded neurons and the stimulation parameters are now explicitly described in the revised Methods section.
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eLife Assessment
Du et al. present a valuable study examining neural activation in medial prefrontal cortex (mPFC) subpopulations projecting to the basolateral amygdala (BLA) and nucleus accumbens (NAc) during behavioral tasks assessing anxiety, social preference, and social dominance. The strength of the evidence linking in vivo neural physiology to behavioral outcomes was considered solid; however, the electrophysiology data and their interpretation were less well received. Overall, the reviewers felt that the revised work provides insight into how distinct mPFC→BLA and mPFC→NAc pathways influence anxiety, exploration, and social behaviors.
-
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 …
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 …
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.
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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 …
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.
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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 …
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.
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eLife Assessment
Du et al. present a valuable study on neural activation in medial prefrontal cortex (mPFC) subpopulations projecting to the basolateral amygdala (BLA) and nucleus accumbens (NAc) during behavioral tasks assessing anxiety, social preference, and social dominance. The study has innovative approaches and solid in vivo calcium imaging data, but the evidence linking neural physiology to behavioral outcomes is incomplete. Addressing these gaps would significantly enhance the understanding of how distinct mPFC→BLA and mPFC→NAc pathways influence anxiety, exploration, and social behaviors.
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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 …
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.
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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 …
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.
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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 …
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.
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