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 have addressed all comments.

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.

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.