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  1. Evaluation Summary:

    The study of cortical regulation of behavior under conflict is novel, timely, and important. It is of broad interest to neuroscientists studying the neural substrates of fear and reward and offers a novel behavioural perspective showing how these opposing motivational states interact to influence behaviour differentially across individuals and how this is regulated by glutamatergic and GABAergic neurons in the prefrontal cortex. The paper uses a variety of cutting-edge tools to dissect the microcircuits of the prefrontal cortex. The claims are generally supported by the data, but some claims comparing individual differences require additional statistical analyses and consideration of potential alternative interpretations for the behavioral phenotypes observed.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  2. Reviewer #3 (Public Review):

    Fernandez-Leon et al. investigate the role of the pre-limbic area (PL) in regulating approach avoidance behavior in situations of learned motivational conflict where animals experience both cues that predict an aversive outcome as well as cues that signal the availability of food. This region has been implicated in threat responding and food seeking separately but has not previously been examined in situations of conflict. The authors employ an individual differences approach, subdividing animals based on their food seeking behavior in the presence of conflicting cues that signal food availability and footshock and use a combination of in vivo recordings and optogenetic manipulations to identify a role for specific cell types in the PL in regulating risky behaviors in aversive contexts. This manuscript adds to the growing literature on neural mechanisms of processing approach-avoidance conflict.

    This work has many strengths. Examining approach and avoidance in a conflict paradigm, rather than separately, provides a more ethological study of the neural basis of these behaviors as, beyond the confines of a laboratory, action selection commonly occurs in the face of multiple competing cues. Subdividing animals into 'pressers' and 'non-pressers' based on individual differences in engagement in food seeking behavior is an excellent strategy to gain insight into the behavioral function of these cells. Recognizing that not engaging in food seeking does not necessarily reflect failure to complete the task but rather a bias toward avoidance behavior is insightful and important. The authors suggest a number of interesting and potentially important differences in PL neural activity between pressers and non-pressers. For example, pressers (i.e. rats that continue to seek food in the presence of an aversive cue) have both more food-cue responsive neurons and greater magnitude of excitatory and inhibitory responses to food-cues, a difference that is sustained when food-cues are presented in the presence of an aversive cue. Pressers and non-pressers also had marked differences in oscillatory frequency, an intriguing finding that warrants further investigation. Optogenetic experiments nicely establish causality with precise temporal resolution.

    The design of the behavioral paradigm somewhat limits the ability the ability to draw certain conclusions. During testing, food-cues were presented discretely while the shock-cue was constant preventing direct comparison of responding to appetitive and aversive cues that would have been highly interesting. Furthermore, during the test session, reward cues are always presented first followed by the addition of the shock-cue. This, and the extended shock-cue presentation under extinction conditions makes it difficult to entirely rule out alternative interpretations for differences between pressers and non-pressers, for example, more rapid extinction of fear memory in pressers than non-pressers. Beyond this, the lack of direct statistical comparison of neural activity in pressers and non-pressers undermines the strength of the central conclusions of this paper.

    The authors hypothesize that stimulating glutamatergic PL neurons decreases signal to noise ratio between cells that are active during food seeking and those that are not, thus resulting in a decrease in food-seeking. This is interesting and plausible proposal to be further explored in future research.

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  3. Reviewer #2 (Public Review):

    This manuscript includes a series of studies to assess the role of prelimbic neurons in mediating behavior during an approach-avoidance conflict task. The authors used a novel task to assess the ability of rats to remember cues previously associated with either reward (food) or threat (footshocks) to make a behavioral decision. In doing so, they uncovered two behavioral phenotypes: "Pressers", who continued to press a lever for food during conflict; and "Non-Pressers", who exhibited a suppression of food-seeking behavior in face of conflict. A combination of optogenetics and single-unit recordings were used to assess the neural mechanisms underlying this individual variability in reward-seeking behavior during conflict. The authors report that increased risk-taking behavior in "Pressers" is associated with reward-cue-elicited responses in the prelimbic cortex and reduced spontaneous activity in prelimbic glutamatergic neurons during conflict. Further, activation of prelimbic glutamatergic, but not GABAergic, neurons attenuated reward-seeking responses selectively in "Pressers"; and inhibition of prelimbic glutamatergic neurons increased reward-approach behavior and decreased freezing behavior during conflict in "Non-Pressers".

    These experiments were well-designed, the methods were appropriate to address the questions at hand, and the manuscript is well-written. The ethologically-relevant approach-avoidance task is novel and will be of interest to the field. In particular, the ability to capture distinct behavioral phenotypes and individual differences using this test will allow further investigation of the neural determinants of reward-seeking and threat-avoiding behavior during conflict.

    As currently presented, there are some concerns regarding the statistical analyses and whether they support all of the authors' claims. As the individual differences component of the manuscript is particularly novel and of interest, it is a bit concerning that these analyses include a sample size of 25 "Pressers" and 7 "Non-Pressers". In relation, it is not clear that the neural responses of these two behavioral phenotypes were ever directly compared. For example, in Figure 2 and Supplementary Figure 2, the area under the curve for neural responses during reward and conflict are presented independently for the two phenotypes and direct comparisons to assess group differences and/or interactions are not apparent. Similarly, it is not clear why data only from "Non-Pressers" is shown in Figure 7, as the methods suggest that both "Pressers" and "Non-Pressers" were used for this experiment. Further, in general, it is difficult to deduce which statistical analyses support the claims made in the manuscript text, as the analyses are only presented in the Figure legends and in Supplemental Table 1 and don't always seem congruent.

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  4. Reviewer #1 (Public Review):

    The paper by Fernandez-Leon examined the role of PL glutamate and GABAergic neurons during a conflict-based behavioural task. The task consisted of lever press during an audio-visual compound in the presence of an aversively conditioned odour. The behavioural data indicated that two cohort of animals were generated - pressers and non-pressers. Pressers continued to press the lever (reward-seek) in the presence of the aversively conditioned odour (albeit to a lesser degree) whereas the non-pressers ceased pressing. Single unit recordings revealed a reduction in the number of food-cue responsive neurons under conflict (compared to no conflict). Different subsets of PL neurons were shown to signal freezing, avoidance and risk-assessment during conflict. The data show reduced spontaneous activity in PL glutamatergic neurons when animals lever press under conflict. Activation of these neurons using ChR2 under the control of the CaMKII promotor attenuated food-seeking behaviour in a neutral context in pressers. Inhibiting the same neurons in non-pressers reduced defensive behaviours often seen to cues conditioned with shock and increased food-based conditioned behaviours.

    The strengths of the paper are numerous and include the novel behavioural design that pits reward up against aversion. Examining the distinct conflict phenotypes throughout the paper was also excellent. The integration of single-cell recordings, LFPs, and optogenetics were considerable strengths allowing to dissect the glutamatergic vs GABAergic microcircuits in the PL during this behaviour. The discussion of the results in the context of the existing literature was excellent.

    Despite the clear strengths of the paper, some weaknesses exist. A closer examination of the single units is warranted. The claim that putative classification of PL neurons into glutamatergic and GABAergic based on waveform and spike timing given the optotagging results seems premature. The optotagging analysis needs additional data including an eYFP control to show what, if any, effect light stimulation alone has on neural responses. Some consideration of whether the two behavioural phenotypes are due to differences under conflict or due to perception is also needed.

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