Parabrachial CGRP neurons modulates conditioned active defensive behavior under a naturalistic threat

  1. Gyeong Hee Pyeon
  2. Hyewon Cho
  3. Byung Min Chung
  4. June-Seek Choi
  5. Yong Sang Jo

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    eLife Assessment

    This important work advances our understanding of parabrachial CGRP threat function. The evidence supporting CGRP aversive outcome signaling is solid, while the evidence for cue signaling and fear behavior generation is incomplete. The work will be of interest to neuroscientists studying defensive behaviors.

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Abstract

Recent studies suggest that calcitonin gene-related peptide (CGRP) neurons in the parabrachial nucleus (PBN) represent aversive information and signal a general alarm to the forebrain. If CGRP neurons serve as a true general alarm, activation of CGRP neurons can trigger either freezing or fleeing defensive behavior, depending on the circumstances. However, the majority of previous findings have reported that CGRP neurons modulate only freezing behavior. Thus, the present study examined the role of CGRP neurons in active defensive behavior, using a predator-like robot programmed to chase mice in fear conditioning. Our electrophysiological results showed that CGRP neurons encoded the intensity of various unconditioned stimuli (US) through different firing durations and amplitudes. Optogenetic and behavioral results revealed that activation of CGRP neurons in the presence of the chasing robot intensified fear memory and significantly elevated conditioned fleeing behavior during recall of an aversive memory. Animals with inactivated CGRP neurons exhibited significantly low levels of fleeing behavior even when the robot was set to be more threatening during conditioning. Our findings expand the known role of CGRP neurons in the PBN as a crucial part of the brain’s alarm system, showing they can regulate not only passive but also active defensive behaviors.

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  1. eLife

    Author response:

    Reviewer #1 (Public Review):

    Summary

    The authors asked if parabrachial CGRP neurons were only necessary for a threat alarm to promote freezing or were necessary for a threat alarm to promote a wider range of defensive behaviors, most prominently flight.

    Major Strengths of Methods and Results

    The authors performed careful single-unit recording and applied rigorous methodologies to optogenetically tag CGRP neurons within the PBN. Careful analyses show that single-units and the wider CGRP neuron population increases firing to a range of unconditioned stimuli. The optogenetic stimulation of experiment 2 was comparatively simpler but achieved its aim of determining the consequence of activating CGRP neurons in the absence of other stimuli. Experiment 3 used a very clever behavioral approach to reveal a setting in which both cue-evoked freezing and flight could be observed. This was done by having the unconditioned stimulus be a "robot" traveling along a circular path at a given speed. Subsequent cue presentation elicited mild flight in controls and optogenetic activation of CGRP neurons significantly boosted this flight response. This demonstrated for the first time that CGRP neuron activation does more than promote freezing. The authors conclude by demonstrating that bidirectional modulation of CGRP neuron activity bidirectionally aTects freezing in a traditional fear conditioning setting and aTects both freezing and flight in a setting in which the robot served as the unconditioned stimulus. Altogether, this is a very strong set of experiments that greatly expand the role of parabrachial CGRP neurons in threat alarm.

    We would like to sincerely thank the reviewer for the positive and insightful comments on our work. We greatly appreciate the acknowledgment of our new behavioral approach, which allowed us to observe a dynamic spectrum of defensive behaviors in animals. Our use of the robot-based paradigm, which enables the observation of both freezing and flight, has been instrumental in expanding our understanding of how parabrachial CGRP neurons modulate diverse threat responses. We are pleased that the reviewer found this methodological innovation to be a valuable contribution to the field.

    Weaknesses

    In all of their conditioning studies the authors did not include a control cue. For example, a sound presented the same number of times but unrelated to US (shock or robot) presentation. This does not detract from their behavioral findings. However, it means the authors do not know if the observed behavior is a consequence of pairing. Or is a behavior that would be observed to any cue played in the setting? This is particularly important for the experiments using the robot US.

    We appreciate the reviewer’s insightful comment regarding the absence of a control cue in our conditioning studies. First, we would like to mention that, in response to the Reviewer 3, we have updated how we present our flight data by following methods from previously published papers (Fadok et al., 2017; Borkar et al., 2024). Instead of counting flight responses, we calculated flight scores as the ratio of the velocity during the CS to the average velocity in the 7 s before the CS on the conditioning day (or 10 s for the retention test). This method better captures both the speed and duration of fleeing during CS. With this updated approach, we observed a significant difference in flight scores between the ChR2 and control groups, even during conditioning, which may partly address the reviewer’s concern about whether the observed behavior is a consequence of CS-US pairing.

    However, we agree with the reviewer that including an unpaired group would provide stronger evidence, and in response, we conducted an additional experiment with an unpaired group. In this unpaired group, the CS was presented the same number of times, but the robot US was delivered randomly within the inter-trial interval. The unpaired group did not exhibit any notable conditioned freezing or flight responses. We believe that this additional experiment, now reflected in Figure 3, further strengthens our conclusion that the fleeing behavior is driven by associative learning between the CS and US, rather than a reaction to the cue itself.

    The authors make claims about the contribution of CGRP neurons to freezing and fleeing behavior, however, all of the optogenetic manipulations are centered on the US presentation period. Presently, the experiments show a role for these neurons in processing aversive outcomes but show little role for these neurons in cue responding or behavior organizing. Claims of contributions to behavior should be substantiated by manipulations targeting the cue period.

    We appreciate the reviewer’s constructive comments. We would like to emphasize that our primary objective in this study was to investigate whether activating parabrachial CGRP neurons—thereby increasing the general alarm signal—would elicit different defensive behaviors beyond passive freezing. To this end, we focused on manipulating CGRP neurons during the US period rather than the cue period.

    Previous studies have shown that CGRP neurons relay US signals, and direct activation of CGRP neurons has been used as the US to successfully induce conditioned freezing responses to the CS during retention tests (Han et al., 2015; Bowen et al., 2020). In our experiments, we also observed that CGRP neurons responded exclusively to the US during conditioning with the robot (Figure 1F), and stimulating these neurons in the absence of any external stimuli elicited strong freezing responses (Figure 2B). These findings, collectively, suggest that activation of CGRP neurons during the CS period would predominantly result in freezing behavior.

    Therefore, we manipulated the activity of CGRP neurons during the US period to examine whether adjusting the perceived threat level through these neurons would result in diverse dfensive behaivors when paired with chasing robot. We observed that enhancing CGRP neuron activity while animals were chased by the robot at 70 cm/s made them react as if chased at a higher speed (90 cm/s), leading to increased fleeing behaviors. While this may not fully address the role of these neurons in cue responding or behavior organizing, we found that silencing CGRP neurons with tetanus toxin (TetTox) abolished fleeing behavior even when animals were chased at high speeds (90 cm/s), which usually elicits fleeing without CGRP manipulation (Figure 5). This supports the conclusion that CGRP neurons are necessary for processing fleeing responses.

    In summary, manipulating CGRP neurons during the US period was essential for effectively investigating their role in adjusting defensive responses, thereby expanding our understanding of their function within the general alarm system. We hope this clarifies our experimental design and addresses the concern the reviewer has raised.

    Appraisal

    The authors achieved their aims and have revealed a much greater role for parabrachial CGRP neurons in threat alarm.

    Discussion

    Understanding neural circuits for threat requires us (as a field) to examine diverse threat settings and behavioral outcomes. A commendable and rigorous aspect of this manuscript was the authors decision to use a new behavioral paradigm and measure multiple behavioral outcomes. Indeed, this manuscript would not have been nearly as impactful had they not done that. This novel behavior was combined with excellent recording and optogenetic manipulations - a standard the field should aspire to. Studies like this are the only way that we as a field will map complete neural circuits for threat.

    We sincerely thank the reviewer for their positive and encouraging comments. We are grateful for the acknowledgment of our efforts in employing a novel behavioral paradigm to study diverse defensive behaviors. We are pleased that our work contributes to advancing the understanding of neural circuits involved in threat responses.

    Reviewer #3 (Public Review):

    Strengths:

    The study used optogenetics together with in vivo electrophysiology to monitor CGRP neuron activity in response to various aversive stimuli including robot chasing to determine whether they encode noxious stimuli diTerentially. The study used an interesting conditioning paradigm to investigate the role of CGRP neurons in the PBN in both freezing and flight behaviors.

    Weakness:

    The major weakness of this study is that the chasing robot threat conditioning model elicits weak unconditioned and conditioned flight responses, making it diTicult to interpret the robustness of the findings. Furthermore, the conclusion that the CGRP neurons are capable of inducing flight is not substantiated by the data. No manipulations are made to influence the flight behavior of the mouse. Instead, the manipulations are designed to alter the intensity of the unconditioned stimulus.

    We sincerely thank the reviewer for the thoughtful and constructive comments on our manuscript. In response to this feedback, we revisited our analysis of the flight responses and compared our methods with those used in previous literatures examining similar behaviors.

    We reviewed a study investigating sex differences in defensive behavior using rats (Gruene et al., 2015). In that study, the CS was presented for 30 s, and active defensive behvaior – referred to as ‘darting’ – was quantified as ‘Dart rate (dart/min)’. This was calculated by doubling the number of darts counted during the 30-s CS presentation to extrapolate to a per-min rate. The highest average dart rate observed was approximatley 1.5. Another relevant studies using mice quantified active defensive behavior by calculating a flight score—the ratio of the average speed during each CS to the average speed during the 10 s pre-CS period (Fadok et al., 2017; Borkar et al., 2024). This method captures multiple aspects of flight behavior during CS presentation, including overall velocity, number of bouts, and duration of fleeing. Moreover, it accounts for each animal’s individual velocity prior to the CS, reflecting how fast the animals were fleeing relative to their baseline activity.

    In our original analysis, we quantified flight responses by counting rapid fleeing movements, defined as movements exceeding 8 cm/s. This approach was consistent with our previous study using the same robot paradigm to observe unique patterns of defensive behavior related to sex differences (Pyeon et al., 2023). Based on our earlier findings, where this approach effectively identified significant differences in defensive behaviors, we believed that this method was appropriate for capturing conditioned flight behavior within our specific experimental context. However, prompted by the reviewer's insightful comments, we recognized that our initial method might not fully capture the robustness of the flight responses. Therefore, we re-analyzed our data using the flight score method described by Fadok and colleagues, which provides a more sensitive measure of fleeing during the CS.

    Re-analyzing our data revealed a more robust flight response than previously reported, demonstrating that additional CGRP neuron stimulation promoted flight behavior in animals during conditioning, addressing the concern that the data did not substantiate the role of CGRP neurons in inducing flight. In addition, we would like to emphasize the findings from our final experiment, where silencing CGRP neurons, even under high-threat conditions (90 cm/s), prevented animals from exhibiting flight responses. This demonstrates that CGRP neurons are necessary in influencing flight responses.

    We have updated all flight data in the manuscript and revised the relevant figures and text accordingly. We appreciate the opportunity to enhance our analysis. The reviewer's insightful observation led us to adopt a better method for quantifying flight behavior, which substantiates our conclusion about the role of CGRP neurons in modulating defensive responses.

    Borkar, C.D., Stelly, C.E., Fu, X., Dorofeikova, M., Le, Q.-S.E., Vutukuri, R., et al. (2024). Top- down control of flight by a non-canonical cortico-amygdala pathway. Nature 625(7996), 743-749.

    Bowen, A.J., Chen, J.Y., Huang, Y.W., Baertsch, N.A., Park, S., and Palmiter, R.D. (2020). Dissociable control of unconditioned responses and associative fear learning by parabrachial CGRP neurons. Elife 9, e59799.

    Fadok, J.P., Krabbe, S., Markovic, M., Courtin, J., Xu, C., Massi, L., et al. (2017). A competitive inhibitory circuit for selection of active and passive fear responses. Nature 542(7639), 96-100.

    Gruene, T.M., Flick, K., Stefano, A., Shea, S.D., and Shansky, R.M. (2015). Sexually divergent expression of active and passive conditioned fear responses in rats. Elife 4, e11352.

    Han, S., Soleiman, M.T., Soden, M.E., Zweifel, L.S., and Palmiter, R.D. (2015). Elucidating an a_ective pain circuit that creates a threat memory. Cell 162(2), 363-374.

    Pyeon, G.H., Lee, J., Jo, Y.S., and Choi, J.-S. (2023). Conditioned flight response in female rats to naturalistic threat is estrous-cycle dependent. Scientific Reports 13(1), 20988.

  2. eLife

    eLife Assessment

    This important work advances our understanding of parabrachial CGRP threat function. The evidence supporting CGRP aversive outcome signaling is solid, while the evidence for cue signaling and fear behavior generation is incomplete. The work will be of interest to neuroscientists studying defensive behaviors.

  3. eLife

    Reviewer #1 (Public Review):

    Summary

    The authors asked if parabrachial CGRP neurons were only necessary for a threat alarm to promote freezing or were necessary for a threat alarm to promote a wider range of defensive behaviors, most prominently flight.

    Major Strengths of Methods and Results

    The authors performed careful single-unit recording and applied rigorous methodologies to optogenetically tag CGRP neurons within the PBN. Careful analyses show that single-units and the wider CGRP neuron population increases firing to a range of unconditioned stimuli. The optogenetic stimulation of experiment 2 was comparatively simpler but achieved its aim of determining the consequence of activating CGRP neurons in the absence of other stimuli. Experiment 3 used a very clever behavioral approach to reveal a setting in which both cue-evoked freezing and flight could be observed. This was done by having the unconditioned stimulus be a "robot" traveling along a circular path at a given speed. Subsequent cue presentation elicited mild flight in controls and optogenetic activation of CGRP neurons significantly boosted this flight response. This demonstrated for the first time that CGRP neuron activation does more than promote freezing. The authors conclude by demonstrating that bidirectional modulation of CGRP neuron activity bidirectionally affects freezing in a traditional fear conditioning setting and affects both freezing and flight in a setting in which the robot served as the unconditioned stimulus. Altogether, this is a very strong set of experiments that greatly expand the role of parabrachial CGRP neurons in threat alarm.

    Weaknesses

    In all of their conditioning studies the authors did not include a control cue. For example, a sound presented the same number of times but unrelated to US (shock or robot) presentation. This does not detract from their behavioral findings. However, it means the authors do not know if the observed behavior is a consequence of pairing. Or is a behavior that would be observed to any cue played in the setting? This is particularly important for the experiments using the robot US.

    The authors make claims about the contribution of CGRP neurons to freezing and fleeing behavior, however, all of the optogenetic manipulations are centered on the US presentation period. Presently, the experiments show a role for these neurons in processing aversive outcomes but show little role for these neurons in cue responding or behavior organizing. Claims of contributions to behavior should be substantiated by manipulations targeting the cue period.

    Appraisal

    The authors achieved their aims and have revealed a much greater role for parabrachial CGRP neurons in threat alarm.

    Discussion

    Understanding neural circuits for threat requires us (as a field) to examine diverse threat settings and behavioral outcomes. A commendable and rigorous aspect of this manuscript was the authors decision to use a new behavioral paradigm and measure multiple behavioral outcomes. Indeed, this manuscript would not have been nearly as impactful had they not done that. This novel behavior was combined with excellent recording and optogenetic manipulations - a standard the field should aspire to. Studies like this are the only way that we as a field will map complete neural circuits for threat.

  4. eLife

    Reviewer #2 (Public Review):

    -Summary of the Authors' Aims:
    The authors aimed to investigate the role of calcitonin gene-related peptide (CGRP) neurons in the parabrachial nucleus (PBN) in modulating defensive behaviors in response to threats. They sought to determine whether these neurons, previously shown to be involved in passive freezing behavior, also play a role in active defensive behaviors, such as fleeing, when faced with imminent threats.

    -Major Strengths and Weaknesses of the Methods and Results:
    The authors utilized an innovative approach by employing a predator-like robot to create a naturalistic threat scenario. This method allowed for a detailed observation of both passive and active defensive behaviors in mice. The combination of electrophysiology, optogenetics, and behavioral analysis provided a comprehensive examination of CGRP neuron activity and its influence on defensive behaviors. The study's strengths lie in its robust methodology, clear results, and the multi-faceted approach that enhances the validity of the findings.

    No notable weakness found.

    -Appraisal of Aims and Results:
    The authors successfully achieved their aims by demonstrating that CGRP neurons in the PBN modulate both passive and active defensive behaviors. The results clearly show that activation of these neurons enhances fear memory and promotes conditioned fleeing behavior, while inhibition reduces these responses. The study provides strong evidence supporting the hypothesis that CGRP neurons act as a comprehensive alarm system in the brain.

    -Impact on the Field and Utility of Methods and Data:
    This work has significant implications for the field of neuroscience, particularly in understanding the neural mechanisms underlying adaptive defensive behaviors. The innovative use of a predator-like robot to simulate naturalistic threats adds ecological validity to the findings and may inspire future studies to adopt similar approaches. The comprehensive analysis of CGRP neuron activity and its role in defensive behaviors provides valuable data that could be useful for researchers studying fear conditioning, neural circuitry, and behavior modulation.

    -Additional Context:
    The study builds on previous research that primarily focused on the role of CGRP neurons in passive defensive responses, such as freezing. By extending this research to include active responses, the authors have provided a more complete picture of the role of these neurons in threat detection and response. The findings highlight the versatility of CGRP neurons in modulating different types of defensive behaviors based on the perceived intensity and immediacy of threats.

    Overall, this manuscript makes a significant contribution to our understanding of the neural basis of defensive behaviors and offers valuable methodological insights for future research in the field.

  5. eLife

    Reviewer #3 (Public Review):

    Strengths:
    The study used optogenetics together with in vivo electrophysiology to monitor CGRP neuron activity in response to various aversive stimuli including robot chasing to determine whether they encode noxious stimuli differentially. The study used an interesting conditioning paradigm to investigate the role of CGRP neurons in the PBN in both freezing and flight behaviors.

    Weakness:
    The major weakness of this study is that the chasing robot threat conditioning model elicits weak unconditioned and conditioned flight responses, making it difficult to interpret the robustness of the findings. Furthermore, the conclusion that the CGRP neurons are capable of inducing flight is not substantiated by the data. No manipulations are made to influence the flight behavior of the mouse. Instead, the manipulations are designed to alter the intensity of the unconditioned stimulus.

  6. Version published to 10.1101/2024.07.23.604768v1 on bioRxiv