A genetically-defined population in the lateral and ventrolateral periaqueductal gray selectively promotes flight to safety
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
The paper presents a tour de force examination of the role of PAG CCK neurons in threat. It is exemplary in the use of a variety of high and low threat tasks along with corresponding behavioural measures, as well as gain and loss of CCK function approaches. The results reported will be of significant benefit for those studying the behavioural and neural mechanisms of learned and unlearned threat, and decision-making in threatening situations.
(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 #1 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Neuroscience (eLife)
Abstract
When encountering external threats, survival depends on the engagement of appropriate defensive reactions to minimize harm. There are major clinical implications for identifying the neural circuitry and activation patterns that produce such defensive reactions, as maladaptive overactivation of these circuits underlies pathological human anxiety and fear responses. A compelling body of work has linked activation of large glutamatergic neuronal populations in the midbrain periaqueductal gray (PAG) to defensive reactions such as freezing, flight and threat-induced analgesia. These pioneering data have firmly established that the overarching functional organization axis of the PAG is along anatomically-defined columnar boundaries. Accordingly, broad activation of the dorsolateral column induces flight, while activation of the lateral or ventrolateral (l and vl) columns induces freezing. However, the PAG contains a diverse arrangement of cell types that vary in neurochemical profile and location. How these cell types contribute to defensive responses remains largely unknown, indicating that targeting sparse, genetically-defined populations can lead to a deeper understanding of how the PAG generates a wide array of behaviors. Though several prior works showed that broad excitation of the lPAG or vlPAG causes freezing, we found that activation of lateral and ventrolateral PAG (l/vlPAG) cholecystokinin-expressing (cck) cells selectively causes flight to safer regions within an environment. Furthermore, inhibition of l/vlPAG-cck cells reduces avoidance of a predatory threat without altering other defensive behaviors like freezing. Lastly, l/vlPAG-cck activity increases away from threat and during movements towards safer locations. In contrast, activating l/vlPAG cells pan-neuronally promoted freezing and these cells were activated near threat. These data underscore the importance of investigating genetically-identified PAG cells. Using this approach, we found a sparse population of cck-expressing l/vlPAG cells that have distinct and opposing function and neural activation motifs compared to the broader local ensemble defined solely by columnar anatomical boundaries. Thus, in addition to the anatomical columnar architecture of the APG, the molecular identity of PAG cells may confer an additional axis of functional organization, revealing unexplored functional heterogeneity.
Article activity feed
-
Author Response
Reviewer #1 (Public Review):
The manuscript contrasts the role of non-specific PAG neurons to PAG cck+ neurons in threat perception using a variety of low and high threat tasks including open field, elevated plus maze, a latency to enter a dark box assay, real-time place preference task, live predatory exposure, fear conditioning. cck neuronal activity in the PAG was examined used gain and loss of function approaches using optogenetics, chemogenetics and fiber photometry. The data show that cck PAG neurons have a dissociable function to the global PAG neuronal response in threatening situations with cck neurons consistently enhancing flight to safety. Specifically, activation of cck PAG neurons decreased time spend in the center of an open field, increased speed and number of corner entries, reduced latency to enter a …
Author Response
Reviewer #1 (Public Review):
The manuscript contrasts the role of non-specific PAG neurons to PAG cck+ neurons in threat perception using a variety of low and high threat tasks including open field, elevated plus maze, a latency to enter a dark box assay, real-time place preference task, live predatory exposure, fear conditioning. cck neuronal activity in the PAG was examined used gain and loss of function approaches using optogenetics, chemogenetics and fiber photometry. The data show that cck PAG neurons have a dissociable function to the global PAG neuronal response in threatening situations with cck neurons consistently enhancing flight to safety. Specifically, activation of cck PAG neurons decreased time spend in the center of an open field, increased speed and number of corner entries, reduced latency to enter a dark box, reduced time in a chamber paired with cck-activation, reduced time spend in the open arms in the elevated plus maze, increasing pupil dilation, enhance avoidance of a live predator, and cck PAG inhibition having the opposite effect to that of activation. Fiber photometry data showed a ramping up upon initiation to escape a live predator, as well as a sustained activity post escape that increased with greater distance from the predator.
The experiments are well executed, the data are clear and convincing. The approach is thorough, and appropriate. The insight is significant and of value beyond the study of threat perception.
No major weaknesses were detected other than the lack of statistical reporting. The conclusion regarding a lack of role for PAG cck neurons in fear learning should be dampened as this would require a more thorough investigation.
We are pleased that the Reviewer finds that our data are “clear”, “convincing” and “appropriate”, and we are heartened that the Reviewer believes the insight provided by this manuscript is “significant.” We apologize for the oversight in statistical reporting. We have now largely expanded our statistical reporting to include complete descriptive statistics throughout the main text and figure legends, including the statistical test used, n’s for each group, and exact p-values. We agree with the Reviewer’s suggestion regarding fear learning and have dampened our interpretation of the involvement of PAG-cck neurons in fear learning in the main text.
Reviewer #2 (Public Review):
This manuscript investigates the role of cck-releasing neurons in ventral regions of the PAG in mediating defensive responses. While prior work in the field has identified columnar organization of mediating defensive responding (i.e., ventral versus dorsal PAG are implicated in freezing and flight, respectively), this work uses several approaches to parse the role of specific cell ensembles in defensive responding.
Through a series of expertly designed studies, the authors have provided compelling evidence that while there are many studies evidencing a role for l/vlPAG in freezing behavior, there may be a more nuanced role for l/vlPAG when considering cell-specific populations. Specifically, cck neurons in l/vlPAG may drive organized escape/ avoidance of threat. Further, activation patterns and behaviors resulting from l/vlPAG manipulation seem to oppose those observed when interrogating l/vlPAG in a non cell type specific manner. These findings underscore the importance of not only neuroanatomical designation of function, but also molecular identification to fully understand the role of defensive response systems.
The identification of a sparse population of cck cells that seem to oppose canonical role for ventral regions of PAG in defensive responding will be of importance to the field. However, there are some caveats that could be made more clear. For example, if l/vlPAG cck neurons initiate escape to safety, it is not fully clear why these cells exhibit greater activity in safe versus threatening locations. One might expect greater activation upon initial escape if this population is the driving force behind the behavior. This raises the possibility that l/vlPAG cck cells coordinate behavioral responses with another population of cells, such as one in the more dorsal regions described by many others to be important for escape and defensive flight. Addressing this would increase the value of the findings presented.
We greatly appreciate the Reviewer’s statement that our “expertly designed studies” “underscore the importance of …. molecular identification to fully understand the role of defensive response systems.”
We believe the Reviewer brings attention to an excellent point regarding why cck+ cells exhibit greater activity in safer locations despite our activation studies driving escape to safety. We interpret these findings in the context of threat avoidance – optogenetic activation induced avoidance of open spaces in low-threat situations and avoidance of a live predator in a high-threat situation. Similarly, we observed enhanced endogenous activity when mice engaged in avoidance from a predator, either by occupying a safe zone most distal from the predator, or actively fleeing from the predator.
The Reviewer is correct to observe that if the population is the driving force behind the behavior, then greater activation should be largely upon the initial portion of escapes. However, we point out that the “safe” zone in the rat assay was not danger-free but was only safer in relation to the threat zone. Even in the safe zone, it is likely that the mouse, in an avoidance state, was motivated to further increase distance from the rat, and this motivation may be related to the increased cck+ activity seen away from the rat.
As this is an extremely important point, we write about this at length in the new Discussion section, ‘l/vlPAG cck cell activity may drive the threat avoidance behavioral state.’
Reviewer #3 (Public Review):
A major role of the PAG in mediating defensive reactions is supported by early microinjection and lesion studies as well as more recent circuit neuroscience studies. By showing that cck neuron activation promoted flight to a burrow, and a global preference for lower threat areas on one hand, and that their activity was correlated with distance to threat on the other, the present study adds to our knowledge of functionally specific circuit elements within the PAG that control different defensive behaviors. Importantly, some of the findings appear contradictory at first glance, and would need to be reconciled via further analyses and/or conceptualization.
The authors systematically performed similar experiments not only with a focus on the l/vlPAG cck neurons, but also on the global neuronal population of the same area. This second aspect mainly recapitulates earlier findings, but most importantly, allows for a direct comparison between a molecularly defined population and the overall neuronal population. This critically highlights that although canonical delineations of the anatomical subregions were adopted based on some neurochemical markers, they do not present an absolute functional and molecular homogeneity, and therefore emphasizes the importance of using specific subpopulations to draw finer conclusions.
The study employs several behavioral paradigms, which are, in the case of the rat exposure test, highly relevant from an ethological point of view, even though conceptual flaws might be present in some aspects of the others.
The experiments, incorporating state-of-the-are techniques are conducted rigorously, and the results are described thoroughly and without overreach. Some analytical approaches need to be described better. Some general points feel like they are not interpreted and conceptualized consequentially enough, including the seemingly contradictory findings. A global picture uniting the different results is missing, which leaves some parts disconnected, yet the data might offer enough elements to develop on that side. The results are well discussed on a higher level and integrated with fitting references for the different aspects of the study, however, the discussion of individual results should be enhanced.
The main weakness of the study is that the perturbational and observational approaches are not easily reconciled. While this is a common phenomenon in circuit research, it hampers a conclusive attribution of the functional role of PAG cck cells and is in contrast to the study's major goal. This discrepancy needs to be resolved both, experimentally and conceptually.
We are pleased that the Reviewer highlights that our “systematically performed” experimentation allows for a “direct comparison between a molecularly defined population and the overall neuronal population.”
We agree with the Reviewer’s assessment that the manuscript would benefit from a “global picture uniting the different results” particularly in the context of the “perturbational and observational” results, and we thankfully use this opportunity to strengthen our manuscript.
To address this need, we have incorporated throughout the main text and in a pointed manner in the Discussion section a global picture that reconciles our perturbational and observational results. We highlight that our results fit with the interpretation that PAG cck+ cells are driving and reflecting threat avoidance states. In this state mice stay away from threat and initiate evasive escape from threat. We show in our perturbational studies that cck+ activity can bidirectionally control threat avoidance measures. In accordance, our observational studies of endogenous cck+ activity show increased activity when mice are engaged in measures of threat avoidance, both by escaping from threat and occupying the zone furthest from threat.
-
Evaluation Summary:
The paper presents a tour de force examination of the role of PAG CCK neurons in threat. It is exemplary in the use of a variety of high and low threat tasks along with corresponding behavioural measures, as well as gain and loss of CCK function approaches. The results reported will be of significant benefit for those studying the behavioural and neural mechanisms of learned and unlearned threat, and decision-making in threatening situations.
(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 #1 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
The manuscript contrasts the role of non-specific PAG neurons to PAG CCK+ neurons in threat perception using a variety of low and high threat tasks including open field, elevated pus maze, a latency to enter a dark box assay, real-time place preference task, live predatory exposure, fear conditioning. CCK neuronal activity in the PAG was examined used gain and loss of function approaches using optogenetics, chemogenetics and fiber photometry. The data show that CCK PAG neurons have a dissociable function to the global PAG neuronal response in threatening situations with CCK neurons consistently enhancing flight to safety. Specifically, activation of CCK PAG neurons decreased time spend in the centre of an open field, increased speed and number of corner entries, reduced latency to enter a dark box, reduced …
Reviewer #1 (Public Review):
The manuscript contrasts the role of non-specific PAG neurons to PAG CCK+ neurons in threat perception using a variety of low and high threat tasks including open field, elevated pus maze, a latency to enter a dark box assay, real-time place preference task, live predatory exposure, fear conditioning. CCK neuronal activity in the PAG was examined used gain and loss of function approaches using optogenetics, chemogenetics and fiber photometry. The data show that CCK PAG neurons have a dissociable function to the global PAG neuronal response in threatening situations with CCK neurons consistently enhancing flight to safety. Specifically, activation of CCK PAG neurons decreased time spend in the centre of an open field, increased speed and number of corner entries, reduced latency to enter a dark box, reduced time in a chamber paired with CCK-activation, reduced time spend in the open arms in the elevated plus maze, increasing pupil dilation, enhanc avoidance of a live predator, and CCK PAG inhibition having the opposite effect to that of activation. Fiber photometry data showed a ramping up upon initiation to escape a live predator, as well as a sustained activity post escape that increased with greater distance from the predator.
The experiments are well executed, the data are clear and convincing. The approach is thorough, and appropriate. The insight is significant and of value beyond the study of threat perception.
No major weaknesses were detected other than the lack of statistical reporting. The conclusion regarding a lack of role for PAG CCK neurons in fear learning should be dampened as this would require a more thorough investigation.
-
Reviewer #2 (Public Review):
This manuscript investigates the role of CCK-releasing neurons in ventral regions of the PAG in mediating defensive responses. While prior work in the field has identified columnar organization of mediating defensive responding (i.e., ventral versus dorsal PAG are implicated in freezing and flight, respectively), this work uses several approaches to parse the role of specific cell ensembles in defensive responding.
Through a series of expertly designed studies, the authors have provided compelling evidence that while there are many studies evidencing a role for l/vlPAG in freezing behavior, there may be a more nuanced role for l/vlPAG when considering cell-specific populations. Specifically, CCK neurons in l/vlPAG may drive organized escape/ avoidance of threat. Further, activation patterns and behaviors …
Reviewer #2 (Public Review):
This manuscript investigates the role of CCK-releasing neurons in ventral regions of the PAG in mediating defensive responses. While prior work in the field has identified columnar organization of mediating defensive responding (i.e., ventral versus dorsal PAG are implicated in freezing and flight, respectively), this work uses several approaches to parse the role of specific cell ensembles in defensive responding.
Through a series of expertly designed studies, the authors have provided compelling evidence that while there are many studies evidencing a role for l/vlPAG in freezing behavior, there may be a more nuanced role for l/vlPAG when considering cell-specific populations. Specifically, CCK neurons in l/vlPAG may drive organized escape/ avoidance of threat. Further, activation patterns and behaviors resulting from l/vlPAG manipulation seem to oppose those observed when interrogating l/vlPAG in a non cell type specific manner. These findings underscore the importance of not only neuroanatomical designation of function, but also molecular identification to fully understand the role of defensive response systems.
The identification of a sparse population of CCK cells that seem to oppose canonical role for ventral regions of PAG in defensive responding will be of importance to the field. However, there are some caveats that could be made more clear. For example, if l/vlPAG CCK neurons initiate escape to safety, it is not fully clear why these cells exhibit greater activity in safe versus threatening locations. One might expect greater activation upon initial escape if this population is the driving force behind the behavior. This raises the possibility that l/vlPAG CCK cells coordinate behavioral responses with another population of cells, such as one in the more dorsal regions described by many others to be important for escape and defensive flight. Addressing this would increase the value of the findings presented.
-
Reviewer #3 (Public Review):
A major role of the PAG in mediating defensive reactions is supported by early microinjection and lesion studies as well as more recent circuit neuroscience studies. By showing that CCK neuron activation promoted flight to a burrow, and a global preference for lower threat areas on one hand, and that their activity was correlated with distance to threat on the other, the present study adds to our knowledge of functionally specific circuit elements within the PAG that control different defensive behaviors. Importantly, some of the findings appear contradictory at first glance, and would need to be reconciled via further analyses and/or conceptualization.
The authors systematically performed similar experiments not only with a focus on the l/vlPAG CCK neurons, but also on the global neuronal population of the …
Reviewer #3 (Public Review):
A major role of the PAG in mediating defensive reactions is supported by early microinjection and lesion studies as well as more recent circuit neuroscience studies. By showing that CCK neuron activation promoted flight to a burrow, and a global preference for lower threat areas on one hand, and that their activity was correlated with distance to threat on the other, the present study adds to our knowledge of functionally specific circuit elements within the PAG that control different defensive behaviors. Importantly, some of the findings appear contradictory at first glance, and would need to be reconciled via further analyses and/or conceptualization.
The authors systematically performed similar experiments not only with a focus on the l/vlPAG CCK neurons, but also on the global neuronal population of the same area. This second aspect mainly recapitulates earlier findings, but most importantly, allows for a direct comparison between a molecularly defined population and the overall neuronal population. This critically highlights that although canonical delineations of the anatomical subregions were adopted based on some neurochemical markers, they do not present an absolute functional and molecular homogeneity, and therefore emphazises the importance of using specific subpopulations to draw finer conclusions.
The study employs several behavioural paradigms, which are, in the case of the rat exposure test, highly relevant from an ethological point of view, even though conceptual flaws might be present in some aspects of the others.
The experiments, incorporating state-of-the-are techniques are conducted rigorously, and the results are described thoroughly and without overreach. Some analytical approaches need to be described better. Some general points feel like they are not interpreted and conceptualized consequentially enough, including the seemingly contradictory findings. A global picture uniting the different results is missing, which leaves some parts disconnected, yet the data might offer enough elements to develop on that side. The results are well discussed on a higher level and integrated with fitting references for the different aspects of the study, however, the discussion of individual results should be enhanced.
The main weakness of the study is that the pertubational and observational approaches are not easily reconciled. While this is a common phenomenon in circuit research, it hampers a conclusive attribution of the functional role of PAG CCK cells and is in contrast to the study's major goal. This discrepancy needs to be resolved both, experimentally and conceptually.
-
