Conditional and unconditional components of aversively motivated freezing, flight and darting in mice
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Evaluation Summary:
This paper will be of interest to neuroscientists, learning theorists and clinicians concerned with factors influencing threat-related response selection relevant to fear vs. panic. The manuscript describes a group of well designed experiments that investigate whether flight-like behaviors reported by other investigators require associative learning in order to occur. The authors demonstrate that non-associative influences can produce strong flight behaviors, but the dataset presented does not eliminate the possibility that associative influences can drive these responses, as well.
(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 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
Fear conditioning is one of the most frequently used laboratory procedures for modeling learning and memory generally, and anxiety disorders in particular. The conditional response (CR) used in the majority of fear conditioning studies in rodents is freezing. Recently, it has been reported that under certain conditions, running, jumping, or darting replaces freezing as the dominant CR. These findings raise both a critical methodological problem and an important theoretical issue. If only freezing is measured but rodents express their learning with a different response, then significant instances of learning, memory, or fear may be missed. In terms of theory, whatever conditions lead to these different behaviors may be a key to how animals transition between different defensive responses and different emotional states. In mice, we replicated these past results but along with several novel control conditions. Contrary to the prior conclusions, running and darting were primarily a result of nonassociative processes and were actually suppressed by associative learning. Darting and flight were taken to be analogous to nonassociative startle or alpha responses that are potentiated by fear. Additionally, associative processes had some impact on the topography of flight behavior. On the other hand, freezing was the purest reflection of associative learning. We also uncovered a rule that describes when these movements replace freezing: when afraid, freeze until there is a sudden novel change in stimulation, then burst into vigorous flight attempts. This rule may also govern the change from fear to panic.
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Author Response
Reviewer #2 (Public Review):
Specific comments/concerns:
- The section relating SCS-flight reactions to alpha responses and fear potentiated startle (FPS) is interesting and potentially important. However, parts of this narrative are unclear. First, FPS has a strong associative component but the flight reaction studied here apparently does not. Second, pairing a tone with shock increases startle reactions preceded by a tone. Here, pairing noise with shock suppresses alpha reactions. Is there evidence that pairing startle-eliciting noises with shock reduces typical startle reactions? Is the issue here that SCS-flight studies are designed poorly to demonstrate the phenomenon (pairing the whole SCS compound with shock vs pairing just the tone with shock then testing noise reactions after a tone)? Lastly, an important …
Author Response
Reviewer #2 (Public Review):
Specific comments/concerns:
- The section relating SCS-flight reactions to alpha responses and fear potentiated startle (FPS) is interesting and potentially important. However, parts of this narrative are unclear. First, FPS has a strong associative component but the flight reaction studied here apparently does not. Second, pairing a tone with shock increases startle reactions preceded by a tone. Here, pairing noise with shock suppresses alpha reactions. Is there evidence that pairing startle-eliciting noises with shock reduces typical startle reactions? Is the issue here that SCS-flight studies are designed poorly to demonstrate the phenomenon (pairing the whole SCS compound with shock vs pairing just the tone with shock then testing noise reactions after a tone)? Lastly, an important experiment by Totty et al (Fig 5) is not discussed. They show that SCS presentations fail to elicit flight reactions in a threatening context (previously paired with unsignaled shocks) unless they were paired with shock in an earlier phase (different context). This seems inconsistent with the FPS interpretation of SCS-flight, since the threatening context should have increased alpha reactions to the novel noise. Along with other control experiments, it also suggests that associative processes related to SCS-shock pairings make a strong contribution to flight. Perhaps there is something unique about compound stimuli paired with shock that cannot be addressed with the simple noise-shock control experiments reported here? This should be discussed in the manuscript.
We thank the reviewer for these insightful suggestions and concerns. First, we have attempted to clarify in the text that for FPS and the cue-elicited activity observed in the experiments presented here, associative fear is necessary for the behavior to be observed; but this does not mean the behavior itself is associative. For example, in FPS, what is measured is a change in the unconditional startle response when the animal is tested in an environment they fear through prior associative learning or after a discrete cue which, through fear conditioning, has acquired fearful properties. If you pair a tone with shock, later presentations of the tone will enhance startle response to a noise (the basic fear-potentiated startle effect). Again, associative fear [to the tone] is necessary, but the potentiated startle is a potentiated unconditional response to a separate noise cue.
Regarding the question of whether pairing a startle-eliciting noise with shock can reduce startle reactions, we do not believe there is prior evidence of this, making this study the first to suggest such a relationship. It is important to realize however, that without fear to the context, the noise stimulus used here supported only very low levels of startle/activity bursts if at all. Yes, we agree that SCS-flight studies are poorly designed to demonstrate the phenomenon of noise-elicited flight, as we show that not only is the compound unnecessary to produce such flight but that SCS studies which do not include standard learning controls for at least pseudoconditioning are unable to determine to what extent any flight is associative vs non-associative.
Finally, we agree that we could have spent more time addressing specific differences and contradictions between our findings and those of Totty et al. We have added one paragraph to the discussion to explicitly address Totty et al. Figure 5 as well as analysis in the results and general text in the discussion to talk about why our observations differ.
- Sex differences have been reported for darting behavior in a Pavlovian paradigm using a single tone CS, but have not been observed in studies using a tone-noise SCS. The combined analyses here (lines 424-429) also finds no sex differences for mice conditioned with a single noise CS. However, the original reports identified only a subset of females that showed prominent darting and stronger shock reactions. Is there any evidence for this Darter vs. Non-darter classification in your dataset? Either way it would be helpful to add graphs illustrating the sex difference analysis that include data points for individuals, at least in supplemental.
We have followed this suggestion and included a Figure 11 which details the sex difference analysis described in the discussion section, including at least a visual analysis of potential sub-populations of darter vs non-darter classification. Based on the criteria set forth in Gruene et al., in our current study with mice, all animals would be classified as darters, and there were no major sex differences—certainly none which suggest greater darting in female mice.
- Lines 432-439: This concluding paragraph is a missed opportunity for a more nuanced discussion of "active vs. passive" defense and perhaps different categories of "flight". The papers cited do not suggest that rats freeze because no other response is available (thought the Blanchards may have said this elsewhere). All the studies investigate CRs in situations where both freezing and locomotor movements are possible. Although it is true that freezing is not the absence of a response, it is the absence of movement. The distinction between movement/ambulation and immobility in threatening situations is important for describing brain circuits of defense and necessary to explain transitions to flight, escape, active avoidance, and even "choosing locations to freeze" by moving down threat gradients. Similar passive vs active terminology goes back at least to Konorski (1967), though "stationary" may be more appropriate than "passive" (Sigmundi, 1987). Related:
-Line 66: "but not activity bursts", Line 77: "Gruene et al suggest that freezing and darting were competing CRs to the same level of threat". Please clarify the Predatory Imminence Theory views on this. If conditioned rats move to the safest spot to freeze (de Oca et al, 2007), is this not an activity burst? Does the velocity of the movement matter? How do these movements relate to the startle-like responses seen at CS onset vs. the more sustained activity reported here for paired groups? de Oca 2007 describes conditioned flight to a familiar enclosure and freezing as compatible post-encounter responses to the same threat, but flight and freezing cannot occur at the same time and must be competitive.
The reviewer brings up important points and important misconceptions that we have [hopefully] addressed in the text. We have added a significant portion to the discussion to detail how we address these concerns. In this section, we attempt to make it much clearer as to our [and the literature’s] position on freezing vs flight vs immobility and active vs passive. Additionally, based both on prior literature and on an analysis of darting topography in our experiments here we suggest that initial ballistic bursts of flight to CS onset are topographically and functionally distinct from subsequent, directed bursts of locomotion which occur later in the CS and may be potentiated by CS-shock pairings. This second burst of locomotion is indeed smaller in our data and we propose that it can be thought of as a behavior that is functionally a part of the freezing suite of behaviors.
- The notion that noise is a (weak) US requires further discussion. Specifically, how do you define a US? And are these properties necessary for the argument that apparent conditioned flight/darting reactions are non-associative startle-like reactions? Freezing goes up when rats experience noise alone trials, but this does not appear to be a result of context conditioning (no BL freezing on day 2 of training). Further, there appears to be no summation once the context is paired with shock (freezing during habituated noise; Exp 4). Noise-elicited freezing appears to sensitize in phase 1 but at the same time darting responses habituate. This pattern is unlike what one might expect for even a very weak shock. One reason this seems important: the paper begins by explaining the challenge posed by the SCS-flight paradigm and the conditioned darting paradigm. However, the studies presented here focus on noise-elicited behavior and imply that similar phenomena occur in the conditioned darting paradigm. The conditioned darting studies all use a pure tone that may not be characterized as a US. Tone-elicited behavior isn't discussed much in the manuscript, but tone-elicited darts in Experiment 2 (pseudoconditioning control) appear lower than those elicited after tone-shock pairings in Experiment 1. So it remains unclear if conditioned darting results from non-associative processes, especially if the tone does not act as a weak US.
We thank this reviewer for pointing out an area that we could be clearer. We have added a section to the discussion to address our views on how stimulus type and properties may impact the behavior observed in these experiments. Generally, our arguments do not require that the CS in question have properties of a US. Additionally, we have added a direct comparison of the Replication and Stimulus Change groups from Experiment 1. While there appear to be slight differences in tone-elicited behavior between these two groups, statistical analyses reveal that while there are general increases in activity bursts in the Stimulus Change Group, these differences were not specific to any particular CS type.
- Baseline data for Darts is missing throughout and should be added to all trial-by-trial graphs. This is important since all phases occurred in same chambers and baseline fear levels could drive darting before stimuli are presented.
We have added baseline darting data to all graphs and found little baseline darting that did not differ between groups and tended to be 0 after Day 1 of each experiment.
- Line 133: "noise was never paired with shock". This is an important point -- but the white noise stimulus contains the tone frequency, and this was paired with shock in the previous phase.
The reviewer brings up an interesting point. While this may be a concern for this experiment, subsequent experiments (2, 3, and 4) all included groups which only received shock with no stimulus-shock pairings.
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Evaluation Summary:
This paper will be of interest to neuroscientists, learning theorists and clinicians concerned with factors influencing threat-related response selection relevant to fear vs. panic. The manuscript describes a group of well designed experiments that investigate whether flight-like behaviors reported by other investigators require associative learning in order to occur. The authors demonstrate that non-associative influences can produce strong flight behaviors, but the dataset presented does not eliminate the possibility that associative influences can drive these responses, as well.
(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 and Reviewer #2 agreed to share their name …
Evaluation Summary:
This paper will be of interest to neuroscientists, learning theorists and clinicians concerned with factors influencing threat-related response selection relevant to fear vs. panic. The manuscript describes a group of well designed experiments that investigate whether flight-like behaviors reported by other investigators require associative learning in order to occur. The authors demonstrate that non-associative influences can produce strong flight behaviors, but the dataset presented does not eliminate the possibility that associative influences can drive these responses, as well.
(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 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Recent studies in rodents suggest the existence of conditioned responses (CRs) to fearful stimuli beyond the classically measured freezing behavior. Here, Trott and colleagues acknowledge that if valid, these results represent an important theoretical issue for prior research using conditioning paradigms. To assess the validity of these prior conclusions, the authors first replicated these behavioral results using similar experimental conditions. Next, they conducted a series of important controls to assess whether these alternative CRs (i.e., darting, flight) are indeed the result of associative processes. In contrast to this model, the authors found that darting and flight where largely the result of nonassociative processes. In general, the experimental design and their accompanying results largely …
Reviewer #1 (Public Review):
Recent studies in rodents suggest the existence of conditioned responses (CRs) to fearful stimuli beyond the classically measured freezing behavior. Here, Trott and colleagues acknowledge that if valid, these results represent an important theoretical issue for prior research using conditioning paradigms. To assess the validity of these prior conclusions, the authors first replicated these behavioral results using similar experimental conditions. Next, they conducted a series of important controls to assess whether these alternative CRs (i.e., darting, flight) are indeed the result of associative processes. In contrast to this model, the authors found that darting and flight where largely the result of nonassociative processes. In general, the experimental design and their accompanying results largely support the authors conclusions. Precisely, they support a model in which what authors refer to as Peak Activity Ratio (PAR) responses are the result from nonassociative processes akin to potentiated startle (or sensitization). As such, in my view, the paper is already quite compelling. It is an excellent study that offers an important contribution to the field. Still, the addition of direct statistical comparisons of data presented in Fig. 2 and S2 and a few clarification statements should aid in the interpretation of the data and the study itself.
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Reviewer #2 (Public Review):
Trott et al examine associative and nonassociative influences on freezing and flight-like responses (locomotion/darting) during and after Pavlovian fear conditioning in mice. Initial experiments use a paradigm popularized recently by Fadok et al 2017 - where a serial conditioned stimulus (SCS) precedes a shock US (tone->noise->shock). They replicate the main result: freezing develops primarily to the tone and activity bursts/darting develop to noise. Control groups demonstrate that noise-elicited flight does not depend on embedding in a compound SCS or pairing noise with shock in the training phase. Velocity measurements during early shock-free tests indicate that vigorous movements occur mostly at noise onset and are bigger when the noise is novel. Since the tone is not necessary for noise-elicited flight, …
Reviewer #2 (Public Review):
Trott et al examine associative and nonassociative influences on freezing and flight-like responses (locomotion/darting) during and after Pavlovian fear conditioning in mice. Initial experiments use a paradigm popularized recently by Fadok et al 2017 - where a serial conditioned stimulus (SCS) precedes a shock US (tone->noise->shock). They replicate the main result: freezing develops primarily to the tone and activity bursts/darting develop to noise. Control groups demonstrate that noise-elicited flight does not depend on embedding in a compound SCS or pairing noise with shock in the training phase. Velocity measurements during early shock-free tests indicate that vigorous movements occur mostly at noise onset and are bigger when the noise is novel. Since the tone is not necessary for noise-elicited flight, all subsequent experiments focus on reactions to noise after treatments in the same context to probe the contribution of associative vs non-associative processes directly. In brief, they find the strongest flight-like reactions when the noise is novel at test and there is a history of shock in the chamber. Habituating the noise or pairing it with shock both blunt subsequent flight reactions to noise onset. Interestingly, less vigorous movements later in the noise cue were potentiated by noise shock pairings (relative to shock-only or unpaired controls), suggesting that a component of the response is associative. Other data suggest that noise behaves as a weak US (disrupts ongoing freezing, repeated presentations support low levels of freezing & darting). The authors conclude that apparent conditioned flight responses are primarily due to non-associative pseudoconditioning/dishabituation of alpha responses to a sudden stimulus change - akin to fear-potentiated startle (FPS). This is an important addition to a recent literature that has generally assumed that active responses after conditioning are associative. The velocity data are especially clear and support the FPS analogy. The conclusions regarding the SCS-flight paradigm are justified by the current data but are partially inconsistent with findings recently published by another lab (Totty et al 2021). It is also unclear if the non-associative processes identified in the present studies apply to conditioned darting studies using a pure tone.
Specific questions/concerns:
1. The section relating SCS-flight reactions to alpha responses and fear potentiated startle (FPS) is interesting and potentially important. However, parts of this narrative are unclear. First, FPS has a strong associative component but the flight reaction studied here apparently does not. Second, pairing a tone with shock increases startle reactions preceded by a tone. Here, pairing noise with shock suppresses alpha reactions. Is there evidence that pairing startle-eliciting noises with shock reduces typical startle reactions? Is the issue here that SCS-flight studies are designed poorly to demonstrate the phenomenon (pairing the whole SCS compound with shock vs pairing just the tone with shock then testing noise reactions after a tone)? Lastly, an important experiment by Totty et al (Fig 5) is not discussed. They show that SCS presentations fail to elicit flight reactions in a threatening context (previously paired with unsignaled shocks) unless they were paired with shock in an earlier phase (different context). This seems inconsistent with the FPS interpretation of SCS-flight, since the threatening context should have increased alpha reactions to the novel noise. Along with other control experiments, it also suggests that associative processes related to SCS-shock pairings make a strong contribution to flight. Perhaps there is something unique about compound stimuli paired with shock that cannot be addressed with the simple noise-shock control experiments reported here? This should be discussed in the manuscript.
2. Sex differences have been reported for darting behavior in a Pavlovian paradigm using a single tone CS, but have not been observed in studies using a tone-noise SCS. The combined analyses here (lines 424-429) also finds no sex differences for mice conditioned with a single noise CS. However, the original reports identified only a subset of females that showed prominent darting and stronger shock reactions. Is there any evidence for this Darter vs. Non-darter classification in your dataset? Either way it would be helpful to add graphs illustrating the sex difference analysis that include data points for individuals, at least in supplemental.
3. Lines 432-439: This concluding paragraph is a missed opportunity for a more nuanced discussion of "active vs. passive" defense and perhaps different categories of "flight". The papers cited do not suggest that rats freeze because no other response is available (thought the Blanchards may have said this elsewhere). All the studies investigate CRs in situations where both freezing and locomotor movements are possible. Although it is true that freezing is not the absence of a response, it is the absence of movement. The distinction between movement/ambulation and immobility in threatening situations is important for describing brain circuits of defense and necessary to explain transitions to flight, escape, active avoidance, and even "choosing locations to freeze" by moving down threat gradients. Similar passive vs active terminology goes back at least to Konorski (1967), though "stationary" may be more appropriate than "passive" (Sigmundi, 1987). Related:
-Line 66: "but not activity bursts", Line 77: "Gruene et al suggest that freezing and darting were competing CRs to the same level of threat". Please clarify the Predatory Imminence Theory views on this. If conditioned rats move to the safest spot to freeze (de Oca et al, 2007), is this not an activity burst? Does the velocity of the movement matter? How do these movements relate to the startle-like responses seen at CS onset vs. the more sustained activity reported here for paired groups? de Oca 2007 describes conditioned flight to a familiar enclosure and freezing as compatible post-encounter responses to the same threat, but flight and freezing cannot occur at the same time and must be competitive.4. The notion that noise is a (weak) US requires further discussion. Specifically, how do you define a US? And are these properties necessary for the argument that apparent conditioned flight/darting reactions are non-associative startle-like reactions? Freezing goes up when rats experience noise alone trials, but this does not appear to be a result of context conditioning (no BL freezing on day 2 of training). Further, there appears to be no summation once the context is paired with shock (freezing during habituated noise; Exp 4). Noise-elicited freezing appears to sensitize in phase 1 but at the same time darting responses habituate. This pattern is unlike what one might expect for even a very weak shock. One reason this seems important: the paper begins by explaining the challenge posed by the SCS-flight paradigm and the conditioned darting paradigm. However, the studies presented here focus on noise-elicited behavior and imply that similar phenomena occur in the conditioned darting paradigm. The conditioned darting studies all use a pure tone that may not be characterized as a US. Tone-elicited behavior isn't discussed much in the manuscript, but tone-elicited darts in Experiment 2 (pseudoconditioning control) appear lower than those elicited after tone-shock pairings in Experiment 1. So it remains unclear if conditioned darting results from non-associative processes, especially if the tone does not act as a weak US.
5. Baseline data for Darts is missing throughout and should be added to all trial-by-trial graphs. This is important since all phases occurred in same chambers and baseline fear levels could drive darting before stimuli are presented.
6. Line 133: "noise was never paired with shock". This is an important point -- but the white noise stimulus contains the tone frequency, and this was paired with shock in the previous phase.
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Reviewer #3 (Public Review):
In this manuscript, Trott et al report a series of elegant experiments designed to parse associative and non-associative influences on escape-like locomotor responses elicited by conditioned stimuli. The work is timely, and the experiments are well designed to assess the contribution of novelty, surprise, and learning to behaviors such as darting and flight. The results paint a complicated picture, the complexities of which need better exploration in the discussion.
The authors' do indeed demonstrate that increased locomotion in response to a white noise can be driven by non-associative factors. This seems particularly true in the 1st and 2nd experiments, in which a novel/surprising serial compound stimulus or a white noise alone triggered a switch from freezing to locomotion more effectively than any …
Reviewer #3 (Public Review):
In this manuscript, Trott et al report a series of elegant experiments designed to parse associative and non-associative influences on escape-like locomotor responses elicited by conditioned stimuli. The work is timely, and the experiments are well designed to assess the contribution of novelty, surprise, and learning to behaviors such as darting and flight. The results paint a complicated picture, the complexities of which need better exploration in the discussion.
The authors' do indeed demonstrate that increased locomotion in response to a white noise can be driven by non-associative factors. This seems particularly true in the 1st and 2nd experiments, in which a novel/surprising serial compound stimulus or a white noise alone triggered a switch from freezing to locomotion more effectively than any associative factor. The picture becomes more complex with experiment 3, however, which compared paired and unpaired groups, as well as a variety of other controls. Contrary to what one might predict on the basis of the first two experiments, the paired group showed less freezing and more darts than the unpaired group (a straightforward interpretation of experiments 1 & 2 would suggest that paired and unpaired should be equal across all measures). Further, the pattern of locomotion driven by non-associative novelty (shock-only group) is very similar to the pattern observed in the unpaired group - a strong spike at the onset of the white noise (alpha response) that returns to near baseline levels by the end of the stimulus. In contrast, the paired group showed a more enduring, multi-peak pattern of elevated locomotion that filled the white noise. The 4th experiment revealed that a locomotor pattern dominated by alpha responses (ie similar to unpaired and shock-only groups in experiment 2) is indeed attenuated by habituation, and thus can be considered non-associative. However, habituation does not seem to have a strong effect on the white noise-filling pattern of locomotion observed in the paired group. Indeed, the descriptions of experiments 3 & 4 in the results section do acknowledge that paired groups produce a particular pattern of locomotor activity/darting that differs from non-associative groups. The discussion, however, does not address this point. Though the paper provides good evidence for the role of non-associative factors in flight-like behavior, it does not totally refute a role for associative factors, as well. Associative factors may not be necessary to produce darting and increased locomotion, but in certain cases they seem sufficient to do so.
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