A fear conditioned cue orchestrates a suite of behaviors in rats
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eLife assessment
This is an important and timely characterization of a diversity of behaviors male and female rats exhibit during the acquisition of Pavlovian fear conditioning in a conditioned suppression procedure. The data are compelling and provide an exhaustive analysis of behavior in a complex associative learning paradigm that blends aversive Pavlovian and appetitive instrumental elements. The generalizability of these findings to other paradigms could be enhanced, however, with the inclusion of tests of cue responses in a neutral environment. These findings are likely to be of interest to those who study fear conditioning and associative learning more broadly in rodents.
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Abstract
Pavlovian fear conditioning has been extensively used to study the behavioral and neural basis of defensive systems. In a typical procedure, a cue is paired with foot shock, and subsequent cue presentation elicits freezing, a behavior theoretically linked to predator detection. Studies have since shown a fear conditioned cue can elicit locomotion, a behavior that – in addition to jumping, and rearing – is theoretically linked to imminent or occurring predation. A criticism of studies observing fear conditioned cue-elicited locomotion is that responding is non-associative. We gave rats Pavlovian fear discrimination over a baseline of reward seeking. TTL-triggered cameras captured 5 behavior frames/s around cue presentation. Experiment 1 examined the emergence of danger-specific behaviors over fear acquisition. Experiment 2 examined the expression of danger-specific behaviors in fear extinction. In total, we scored 112,000 frames for nine discrete behavior categories. Temporal ethograms show that during acquisition, a fear conditioned cue suppresses reward seeking and elicits freezing, but also elicits locomotion, jumping, and rearing – all of which are maximal when foot shock is imminent. During extinction, a fear conditioned cue most prominently suppresses reward seeking, and elicits locomotion that is timed to shock delivery. The independent expression of these behaviors in both experiments reveals a fear conditioned cue to orchestrate a temporally organized suite of behaviors.
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eLife assessment
This is an important and timely characterization of a diversity of behaviors male and female rats exhibit during the acquisition of Pavlovian fear conditioning in a conditioned suppression procedure. The data are compelling and provide an exhaustive analysis of behavior in a complex associative learning paradigm that blends aversive Pavlovian and appetitive instrumental elements. The generalizability of these findings to other paradigms could be enhanced, however, with the inclusion of tests of cue responses in a neutral environment. These findings are likely to be of interest to those who study fear conditioning and associative learning more broadly in rodents.
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Reviewer #1 (Public Review):
This report describes an exhaustive analysis of behavior in a complex associative learning paradigm that blends aversive Pavlovian and appetitive instrumental elements. The hand-scoring technique is rigorous and documented to a greater degree than what is typically reported in papers using human raters to quantify animal behavior. Near-complete ethograms offer a novel, high-resolution look at how aversive cues exert distinct effects on appetitive and aversive behavior.
From the perspective of the rodent subject, there is quite a lot going on in the experimental chamber in this study. It's an environment in which appetitive instrumental action is set against multiple predictive cues signaling differing degrees of danger and safety. The test is fully on-baseline, occurring in the same place as training. The …
Reviewer #1 (Public Review):
This report describes an exhaustive analysis of behavior in a complex associative learning paradigm that blends aversive Pavlovian and appetitive instrumental elements. The hand-scoring technique is rigorous and documented to a greater degree than what is typically reported in papers using human raters to quantify animal behavior. Near-complete ethograms offer a novel, high-resolution look at how aversive cues exert distinct effects on appetitive and aversive behavior.
From the perspective of the rodent subject, there is quite a lot going on in the experimental chamber in this study. It's an environment in which appetitive instrumental action is set against multiple predictive cues signaling differing degrees of danger and safety. The test is fully on-baseline, occurring in the same place as training. The rich web of associations formed has a predictably complex influence on behavior. The authors contrast this complexity with much of the rest of the literature, in which freezing is reported to predominate when an aversive CS is presented. Indeed, most conventional studies of aversive associative learning train subjects on a single tone-shock association and test in a neutral context. The contrast between the common approach and the one taken by the authors suggests questions central to understanding the current report. Does being tested in an associatively complex context promote the pattern of behaviors that the authors observe? Or is it a question of learning history - would, following this kind of complex training, an off-baseline test in a neutral environment, produce the same suite of outcomes in response to the danger cue? Answers to these questions would go some distance toward nesting this paper in a wider body of knowledge about defensive reactions to aversive conditioned stimuli. Data speaking to these issues would also increase the work's impact by demonstrating the way in which a given response can be modulated by other learning.
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Reviewer #2 (Public Review):
This is an important and timely characterization of a diversity of behaviors male and female rats exhibit during the acquisition of Pavlovian fear conditioning in a conditioned suppression procedure. Using hand-scored video analysis and ethogram of nine different behaviors, the authors report that auditory conditioned stimuli that predict shock with high certainty evoke not only freezing, but a variety of other behaviors including locomotion, jumping, and rearing (in addition to suppressing reward-seeking). Auditory stimuli that were followed by shock on only some trials (uncertainty condition), were less likely to evoke freezing and did not lead to a suppression of port/cup-directed behaviors (reward seeking). There were subtle sex differences in the temporal profile of freezing behavior, but not in the …
Reviewer #2 (Public Review):
This is an important and timely characterization of a diversity of behaviors male and female rats exhibit during the acquisition of Pavlovian fear conditioning in a conditioned suppression procedure. Using hand-scored video analysis and ethogram of nine different behaviors, the authors report that auditory conditioned stimuli that predict shock with high certainty evoke not only freezing, but a variety of other behaviors including locomotion, jumping, and rearing (in addition to suppressing reward-seeking). Auditory stimuli that were followed by shock on only some trials (uncertainty condition), were less likely to evoke freezing and did not lead to a suppression of port/cup-directed behaviors (reward seeking). There were subtle sex differences in the temporal profile of freezing behavior, but not in the properties of the other behaviors under study.
Ultimately, these findings point to the importance of task variables (eg., reward seeking in a conditioned suppression procedure) and shock probability in shaping an animal's defense repertoire under threat.
An important factor that this work does not resolve is how the magnitude of the threat/shock (and presumably the state of fear that it engenders) influences an animal's defensive topography. This report used a modest/weak footshock intensity that supported very low levels of tone-elicited freezing (<20%) - a stark contrast to the extant fear conditioning literature that typically reports much higher levels of freezing behavior.
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Reviewer #3 (Public Review):
The authors' goal was to explore if there were fear behaviors expressed to a conditioned fear cue other than freezing and how the timing of these behaviors may change across a discrete conditioned cue. Three separate cues representing danger (1.0 footshock probability), safety (0 footshock probability), and uncertainty (0.25 footshock probability) were used against a backdrop of operant nosepoke responding for reward in male and female Long Evans rats. All behaviors were recorded with a frame capture of 5 frames per second and manually scored afterwards blindly for one of ten behaviors.
Analyzing the repertoire of possible behaviors, beyond freezing, across a 10s conditioned cue that may be perceived as dangerous, uncertain, or safe is a strength of the study. Displaying the possible behaviors stacked across …
Reviewer #3 (Public Review):
The authors' goal was to explore if there were fear behaviors expressed to a conditioned fear cue other than freezing and how the timing of these behaviors may change across a discrete conditioned cue. Three separate cues representing danger (1.0 footshock probability), safety (0 footshock probability), and uncertainty (0.25 footshock probability) were used against a backdrop of operant nosepoke responding for reward in male and female Long Evans rats. All behaviors were recorded with a frame capture of 5 frames per second and manually scored afterwards blindly for one of ten behaviors.
Analyzing the repertoire of possible behaviors, beyond freezing, across a 10s conditioned cue that may be perceived as dangerous, uncertain, or safe is a strength of the study. Displaying the possible behaviors stacked across the 10s, second by second, instead of a bulk 10s average of each type of behavior highlights the dynamic nature of the defensive behaviors expressed across time. It is unclear though why the 2s post-cue were not included since the footshock was not administered until 2s after cue offset. Given their argument of defensive behaviors being adjusted as the threat becomes more imminent, this 2s period would appear to be a valuable interval for their analyses and argument.
The authors emphasize the ethology of their findings but they also acknowledge that their findings do not agree with the majority of rodent fear conditioning papers reporting upwards of 80% freezing across a cue. Since these differences could be due to a myriad of experimental differences such as cue length, cue modality, number and strength of shocks, etc, it is difficult to extrapolate and apply the reported findings to potentially broader conditions; e.g. cues that are not 10s, non-rodent species, food-restricted vs not food-restricted, an environment that is not a small, enclosed box, etc. In the end, while additional defensive behaviors were reported in response to a danger cue, the predominant behavior still appeared to be freezing, although there were interesting differences noted between males and females in that females appeared to display most of their freezing early in the cue while males express a more sustained freezing response across the cue.
This work could certainly inspire other labs to approach their video analyses in a similar fashion and, although not discussed in the paper, could potentially be interesting to also look at individual differences across ethograms, instead of the grouped data presented across the 12 males versus 12 females as shown here. These could then be used before or after a manipulation and used to try to predict how an animal may respond to a certain event or manipulation.
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