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SCIENTIST AT WORK: Joseph LeDoux

Using Rats to Trace Anatomy of Fear, Biology of Emotion

By Sandra Blakeslee

Nov. 5, 1996

THE realm of emotion and feeling is a treacherous one for science. Rage, lust, envy and shame churn in the human psyche. Yearning, disappointment and fear mingle with conscious thought, sway decisions and then recede like phantoms.

Writers, psychoanalysts and psychologists may try to sort out the interplay of cognition and desire, of thought and compulsion. But understanding the origin and architecture of human emotions in the brain is another matter altogether. What is the key to linking emotions to activity in the cells of the brain?

Rats -- better yet, frightened rats -- are the key, says Dr. Joseph LeDoux, a 46-year-old neuroscientist at New York University who pioneered the study of emotions as biological phenomena.

''You start with something you can study,'' Dr. LeDoux said in a recent interview. Like most animals, rats exhibit fear, an emotion that may help creatures escape from predators. In experiments over the last 15 years, Dr. LeDoux has traced fear inside the rat's brain -- from the first sounds of danger detected by the outer ear to inner brain circuits that cause the animal either to freeze or to run for its life.

Using this strategy, Dr. LeDoux has given researchers the first real glimpse into the neuroanatomy of an emotion. And though the work has been done in rats, Dr. Ledoux says the findings also apply to humans, providing insights into why it is so difficult to control emotions with rational, conscious thought.

One of the biggest surprises from Dr. LeDoux's work is that there may be no such thing as the limbic system -- a brain structure that has been supposed to underlie emotion and motivation. All students are taught about the limbic system, Dr. LeDoux said, ''but in my opinion, it's no longer a valid concept.''

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Observational fear learning in rats: role of trait anxiety and ultrasonic vocalization.

1. Introduction

2. materials and methods, 2.1. animals and housing, 2.2. apparatus, 2.3. behavioral procedure, 2.3.1. experiment 1: role of trait anxiety, 2.3.2. experiment 2: role of ultrasonic vocalization, 2.4. offline analyses of behavior and ultrasonic vocalization, 2.5. descriptive and analytical statistics, 3.1. experiment 1: role of trait anxiety, 3.2. experiment 2: role of ultrasonic vocalization, 4. discussion, 5. conclusions, author contributions, institutional review board statement, informed consent statement, data availability statement, acknowledgments, conflicts of interest.

Blanchard, R.J.; Blanchard, D.C.; Rodgers, J.; Weiss, S.M. The characterization and modelling of antipredator defensive behavior. Neurosci. Biobehav. Rev. 1990 , 14 , 463–472. [ Google Scholar ] [ CrossRef ]
Blanchard, R.J.; Blanchard, D.C. An ethoexperimental analysis of defense, fear, and anxiety. In Anxiety ; McNaughton, N., Andrews, G., Eds.; University of Otago Press: Dunedin, New Zealand, 1990; pp. 124–133. [ Google Scholar ]
Blanchard, R.J.; Blanchard, D.C. Crouching as an index of fear. J. Comp. Physiol. Psychol. 1969 , 67 , 370–375. [ Google Scholar ] [ CrossRef ]
Blanchard, R.J.; Blanchard, D.C.; Agullana, R.; Weiss, S.M. Twenty-two kHz alarm cries to presentation of a predator, by laboratory rats living in visible burrow systems. Physiol. Behav. 1991 , 50 , 967–972. [ Google Scholar ] [ CrossRef ]
Brudzynski, S.M. Pharmacological and behavioral characteristics of 22 kHz alarm calls in rats. Neurosci. Biobehav. Rev. 2001 , 25 , 611–617. [ Google Scholar ] [ CrossRef ]
Brudzynski, S.M.; Bihari, F.; Ociepa, D.; Fu, X.W. Analysis of 22 kHz ultrasonic vocalization in laboratory rats: Long and short calls. Physiol. Behav. 1993 , 54 , 215–221. [ Google Scholar ] [ CrossRef ]
Wöhr, M.; Borta, A.; Schwarting, R.K.W. Overt behavior and ultrasonic vocalization in a fear conditioning paradigm: A dose-response study in the rat. Neurobiol. Learn. Mem. 2005 , 84 , 228–240. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Wöhr, M.; Schwarting, R.K.W. Affective communication in rodents: Ultrasonic vocalizations as a tool for research on emotion and motivation. Cell Tiss. Res. 2013 , 354 , 81–97. [ Google Scholar ] [ CrossRef ]
Brudzynski, S.M. Ethotransmission: Communication of emotional states through ultrasonic vocalization in rats. Curr. Opin. Neurobiol. 2013 , 23 , 310–317. [ Google Scholar ] [ CrossRef ]
Brudzynski, S.M.; Chiu, E.M. Behavioural responses of laboratory rats to playback of 22 kHz ultrasonic calls. Physiol. Behav. 1995 , 57 , 1039–1044. [ Google Scholar ] [ CrossRef ]
Wöhr, M.; Schwarting, R.K.W. Ultrasonic communication in rats: Can playback of 50-kHz calls induce approach behavior? PLoS ONE 2007 , 2 , e1365. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Sales, G.D. The effect of 22 kHz calls and artificial 38 kHz signals on activity in rats. Behav. Proc. 1991 , 24 , 83–93. [ Google Scholar ] [ CrossRef ]
Endres, T.; Widmann, K.; Fendt, M. Are rats predisposed to learn 22 kHz calls as danger-predicting signals? Behav. Brain Res. 2007 , 185 , 69–75. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Blanchard, D.C.; Blanchard, R.J. Defensive behaviors, fear, and anxiety. In Handbook of Behavioral Neuroscience ; Blanchard, R.J., Blanchard, D.C., Griebel, G., Nutt, D.J., Eds.; Elsevier: Amsterdam, The Netherlands, 2008; Volume 17, pp. 63–79. [ Google Scholar ]
Fanselow, M.S. The role of learning in threat imminence and defensive behaviors. Curr. Opin. Behav. Sci. 2018 , 24 , 44–49. [ Google Scholar ] [ CrossRef ]
LeDoux, J.E. Coming to terms with fear. Proc. Natl. Acad. Sci. USA 2014 , 111 , 2871–2878. [ Google Scholar ] [ CrossRef ] [ Green Version ]
LeDoux, J.E. Emotion circuits in the brain. Annu. Rev. Neurosci. 2000 , 23 , 155–184. [ Google Scholar ] [ CrossRef ]
Fendt, M.; Fanselow, M.S. The neuroanatomical and neurochemical basis of conditioned fear. Neurosci. Biobehav. Rev. 1999 , 23 , 743–760. [ Google Scholar ] [ CrossRef ]
Maren, S. Neurobiology of Pavlovian fear conditioning. Annu. Rev. Neurosci. 2001 , 24 , 897–931. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Pape, H.C.; Pare, D. Plastic synaptic networks of the amygdala for the acquisition, expression, and extinction of conditioned fear. Physiol. Rev. 2010 , 90 , 419–463. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Lissek, S.; Rabin, S.; Heller, R.E.; Lukenbaugh, D.; Geraci, M.; Pine, D.S.; Grillon, C. Overgeneralization of conditioned fear as a pathogenic marker of panic disorder. Am. J. Psychiatry 2010 , 167 , 47–55. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Lissek, S.; Kaczkurkin, A.N.; Rabin, S.; Geraci, M.; Pine, D.S.; Grillon, C. Generalized anxiety disorder is associated with overgeneralization of classically conditioned fear. Biol. Psychiatry 2014 , 75 , 909–915. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
Rabinak, C.A.; Mori, S.; Lyons, M.; Milad, M.R.; Phan, K.L. Acquisition of CS-US contingencies during Pavlovian fear conditioning and extinction in social anxiety disorder and posttraumatic stress disorder. J. Affect. Dis. 2017 , 207 , 76–85. [ Google Scholar ] [ CrossRef ]
Grillon, C.; Ameli, R.; Foot, M.; Davis, M. Fear-potentiated startle: Relationship to the level of state/trait anxiety in healthy subjects. Biol. Psychiatry 1993 , 33 , 566–574. [ Google Scholar ] [ CrossRef ]
Gazendam, F.J.; Kamphuis, J.H.; Kindt, M. Deficient safety learning characterizes high trait anxious individuals. Biol. Psychol. 2013 , 92 , 342–352. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Haaker, J.; Lonsdorf, T.B.; Schumann, D.; Menz, M.; Brassen, S.; Bunzeck, N.; Gamer, M.; Kalisch, R. Deficient inhibitory processing in trait anxiety: Evidence from context-dependent fear learning, extinction recall and renewal. Biol. Psychol. 2015 , 111 , 65–72. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Ilse, A.; Prameswari, V.; Kahl, E.; Fendt, M. The role of trait anxiety in associative learning during and after an aversive event. Learn. Mem. 2019 , 26 , 56–59. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Muigg, P.; Hetzenauer, A.; Hauer, G.; Hauschild, M.; Gaburro, S.; Frank, E.; Landgraf, R.; Singewald, N. Impaired extinction of learned fear in rats selectively bred for high anxiety—Evidence of altered neuronal processing in prefrontal-amygdala pathways. Eur. J. Neurosci. 2008 , 28 , 2299–2309. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Atsak, P.; Orre, M.; Bakker, P.; Cerliani, L.; Roozendaal, B.; Gazzola, V.; Moita, M.; Keysers, C. Experience modulates vicarious freezing in rats: A model for empathy. PLoS ONE 2011 , 6 , e21855. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Bruchey, A.K.; Jones, C.E.; Monfils, M.H. Fear conditioning by-proxy: Social transmission of fear during memory retrieval. Behav. Brain Res. 2010 , 214 , 80–84. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Jones, C.E.; Monfils, M.H. Dominance status predicts social fear transmission in laboratory rats. Anim. Cogn. 2016 , 19 , 1051–1069. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Jones, C.E.; Riha, P.D.; Gore, A.C.; Monfils, M.H. Social transmission of Pavlovian fear: Fear-conditioning by-proxy in related female rats. Anim. Cogn. 2014 , 17 , 827–834. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Jones, C.E.; Agee, L.; Monfils, M.H. Fear conditioning by proxy: Social transmission of fear between interacting conspecifics. Curr. Protoc. Neurosci. 2018 , 83 , e43. [ Google Scholar ] [ CrossRef ]
Wöhr, M. Ultrasonic communication in rats: Appetitive 50-kHz ultrasonic vocalizations as social contact calls. Behav. Ecol. Sociobiol. 2017 , 72 , 14. [ Google Scholar ] [ CrossRef ]
Brudzynski, S.M.; Pniak, A. Social contacts and production of 50-kHz short ultrasonic calls in adult rats. J. Comp. Psychol. 2002 , 116 , 73–82. [ Google Scholar ] [ CrossRef ]
Mulvihill, K.G.; Brudzynski, S.M. Non-pharmacological induction of rat 50 kHz ultrasonic vocalization: Social and non-social contexts differentially induce 50 kHz call subtypes. Physiol. Behav. 2018 , 196 , 200–207. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Kelly, M.M.; Forsyth, J.P. Associations between emotional avoidance, anxiety sensitivity, and reactions to an observational fear challenge procedure. Behav. Res. Ther. 2009 , 47 , 331–338. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Misane, I.; Tovote, P.; Meyer, M.; Spiess, J.; Ogren, S.O.; Stiedl, O. Time-dependent involvement of the dorsal hippocampus in trace fear conditioning in mice. Hippocampus 2005 , 15 , 418–426. [ Google Scholar ] [ CrossRef ]
Wright, J.M.; Gourdon, J.C.; Clarke, P.B. Identification of multiple call categories within the rich repertoire of adult rat 50-kHz ultrasonic vocalizations: Effects of amphetamine and social context. Psychopharmacology 2010 , 211 , 1–13. [ Google Scholar ] [ CrossRef ]
Jeon, D.; Kim, S.; Chetana, M.; Jo, D.; Ruley, H.E.; Lin, S.Y.; Rabah, D.; Kinet, J.P.; Shin, H.S. Observational fear learning involves affective pain system and Ca v 1.2 Ca 2+ channels in ACC. Nat. Neurosci. 2010 , 13 , 482–488. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Kim, S.; Mátyás, F.; Lee, S.; Acsády, L.; Shin, H.S. Lateralization of observational fear learning at the cortical but not thalamic level in mice. Proc. Natl. Acad. Sci. USA 2012 , 109 , 15497–15501. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Knapska, E.; Mikosz, M.; Werka, T.; Maren, S. Social modulation of learning in rats. Learn. Mem. 2010 , 17 , 35–42. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Keum, S.; Shin, H.S. Neural basis of observational fear learning: A potential model of affective empathy. Neuron 2019 , 104 , 78–86. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Debiec, J.; Olsson, A. Social fear learning: From animal models to human function. Trends Cogn. Sci. 2017 , 21 , 546–555. [ Google Scholar ] [ CrossRef ]
Sartori, S.B.; Hauschild, M.; Bunck, M.; Gaburro, S.; Landgraf, R.; Singewald, N. Enhanced fear expression in a psychopathological mouse model of trait anxiety: Pharmacological interventions. PLoS ONE 2011 , 6 , e16849. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
Nobre, M.J.; Cabral, A.; Brandão, M.L. GABAergic regulation of auditory sensory gating in low- and high-anxiety rats submitted to a fear conditioning procedure. Neuroscience 2010 , 171 , 1152–1163. [ Google Scholar ] [ CrossRef ]
Silvers, J.A.; Callaghan, B.L.; VanTieghem, M.; Choy, T.; O’Sullivan, K.; Tottenham, N. An exploration of amygdala-prefrontal mechanisms in the intergenerational transmission of learned fear. Dev. Sci. 2020 , e13056. [ Google Scholar ] [ CrossRef ]
Surcinelli, P.; Codispoti, M.; Montebarocci, O.; Rossi, N.; Baldaro, B. Facial emotion recognition in trait anxiety. J. Anxiety Dis. 2006 , 20 , 110–117. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Cooper, R.M.; Rowe, A.C.; Penton-Voak, I.S. The role of trait anxiety in the recognition of emotional facial expressions. J. Anxiety Dis. 2008 , 22 , 1120–1127. [ Google Scholar ] [ CrossRef ]
Schwarting, R.K.W.; Wöhr, M. On the relationships between ultrasonic calling and anxiety-related behavior in rats. Braz. J. Med. Biol. Res. 2012 , 45 , 337–348. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Wöhr, M.; Schwarting, R.K.W. Ultrasonic calling during fear conditioning in the rat: No evidence for an audience effect. Anim. Behav. 2008 , 76 , 749–760. [ Google Scholar ] [ CrossRef ]
Davitz, J.R.; Mason, D.J. Socially facilitated reduction of a fear response in rats. J. Comp. Physiol. Psychol. 1955 , 48 , 149–151. [ Google Scholar ] [ CrossRef ]
Kiyokawa, Y.; Hennessy, M.B. Comparative studies of social buffering: A consideration of approaches, terminology, and pitfalls. Neurosci. Biobehav. Rev. 2018 , 86 , 131–141. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Fendt, M.; Brosch, M.; Wernecke, K.E.A.; Willadsen, M.; Wöhr, M. Predator odour but not TMT induces 22-kHz ultrasonic vocalizations in rats that lead to defensive behaviours in conspecifics upon replay. Sci. Rep. 2018 , 8 , 11041. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Knutson, B.; Burgdorf, J.; Panksepp, J. Ultrasonic Vocalizations as indices of affective states in rats. Psychol. Bull. 2002 , 128 , 961–977. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Burgdorf, J.; Kroes, R.A.; Moskal, J.R.; Pfaus, J.G.; Brudzynski, S.M.; Panksepp, J. Ultrasonic vocalizations of rats ( Rattus norvegicus ) during mating, play, and aggression: Behavioral concomitants, relationship to reward, and self-administration of playback. J. Comp. Psychol. 2008 , 122 , 357–367. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
Burke, C.J.; Kisko, T.M.; Swiftwolfe, H.; Pellis, S.M.; Euston, D.R. Specific 50-kHz vocalizations are tightly linked to particular types of behavior in juvenile rats anticipating play. PLoS ONE 2017 , 12 , e0175841. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
Himmler, B.T.; Kisko, T.M.; Euston, D.R.; Kolb, B.; Pellis, S.M. Are 50-kHz calls used as play signals in the playful interactions of rats? I. Evidence from the timing and context of their use. Behav. Proc. 2014 , 106 , 60–66. [ Google Scholar ] [ CrossRef ] [ PubMed ]
Defensor, E.B.; Corley, M.J.; Blanchard, R.J.; Blanchard, D.C. Facial expressions of mice in aggressive and fearful contexts. Physiol. Behav. 2012 , 107 , 680–685. [ Google Scholar ] [ CrossRef ]
Inagaki, H.; Kiyokawa, Y.; Tamogami, S.; Watanabe, H.; Takeuchi, Y.; Mori, Y. Identification of a pheromone that increases anxiety in rats. Proc. Natl. Acad. Sci. USA 2014 , 111 , 18751–18756. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Breitfeld, T.; Bruning, J.E.A.; Inagaki, H.; Takeuchi, Y.; Kiyokawa, Y.; Fendt, M. Temporary inactivation of the anterior part of the bed nucleus of the stria terminalis blocks alarm pheromone-induced defensive behavior in rats. Front. Neurosci. 2015 , 9 , 321. [ Google Scholar ] [ CrossRef ] [ Green Version ]
Kiyokawa, Y.; Shimozuru, M.; Kikusui, T.; Takeuchi, Y.; Mori, Y. Alarm pheromone increases defensive and risk assessment behaviors in male rats. Physiol. Behav. 2006 , 87 , 383–387. [ Google Scholar ] [ CrossRef ] [ Green Version ]

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Fendt, M.; Gonzalez-Guerrero, C.P.; Kahl, E. Observational Fear Learning in Rats: Role of Trait Anxiety and Ultrasonic Vocalization. Brain Sci. 2021 , 11 , 423. https://doi.org/10.3390/brainsci11040423

Fendt M, Gonzalez-Guerrero CP, Kahl E. Observational Fear Learning in Rats: Role of Trait Anxiety and Ultrasonic Vocalization. Brain Sciences . 2021; 11(4):423. https://doi.org/10.3390/brainsci11040423

Fendt, Markus, Claudia Paulina Gonzalez-Guerrero, and Evelyn Kahl. 2021. "Observational Fear Learning in Rats: Role of Trait Anxiety and Ultrasonic Vocalization" Brain Sciences 11, no. 4: 423. https://doi.org/10.3390/brainsci11040423

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The conditions that regulate formation of a false fear memory in rats

Affiliations.

1 School of Psychology, University of New South Wales, Sydney, NSW 2052, Australia. Electronic address: [email protected].
2 School of Psychology, University of New South Wales, Sydney, NSW 2052, Australia. Electronic address: [email protected].
3 School of Psychology, University of New South Wales, Sydney, NSW 2052, Australia. Electronic address: [email protected].
4 School of Psychology, University of New South Wales, Sydney, NSW 2052, Australia. Electronic address: [email protected].
5 School of Psychology, University of New South Wales, Sydney, NSW 2052, Australia. Electronic address: [email protected].
6 School of Psychology, University of New South Wales, Sydney, NSW 2052, Australia. Electronic address: [email protected].
PMID: 30359728
DOI: 10.1016/j.nlm.2018.10.009

People and animals sometimes associate events that never occurred together. These false memories can have disastrous consequences, yet little is known about the conditions under which they form. In four experiments, we investigated how rats learn to fear a context in which they have never experienced danger (i.e., how they form a false context fear memory). In each experiment, rats were pre-exposed to a context on day 1, shocked in a similar-but-different context on day 2, and tested in the pre-exposed or explicitly-conditioned context on day 3. The results revealed that: (1) the true memory of the explicitly-conditioned context and false memory of the pre-exposed context develop simultaneously and independently; and (2) the conditions of pre-exposure on day 1 and time of shock exposure on day 2 interact to determine the strength of the false memory. These findings are anticipated by a recent computational model, the Bayesian Context Fear Algorithm/Automaton (BACON; Krasne, Cushman, & Fanselow, 2015). They are discussed in relation to this model and more general theories of context learning.

Keywords: Context fear conditioning; False memory; Mediated conditioning; Memory.

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Research Article
Neuroscience

A fear conditioned cue orchestrates a suite of behaviors in rats

Nicholas T Gordon
Aleah M DuBois
Christa B Michel
Katherine E Hanrahan
David C Williams
Stefano Anzellotti
Department of Psychology and Neuroscience, Boston College, United States ;
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Michael A McDannald
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Elife digest, introduction, materials and methods, data availability, article and author information.

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.

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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Knowing that an animal is fearful is crucial for many psychology and neuroscience studies. For instance, this knowledge allows researchers to examine the brain pathways involved in processing and responding to fear.

Typically, researchers consider that a rodent is experiencing fear if it ‘freezes’ – a response which, in the wild, helps to evade detection by predators. In Pavlovian fear conditioning experiments, for example, rats and mice freeze when exposed to a stimulus (often a specific sound) previously associated with unpleasant sensations. However, rodents can also respond more actively to threats, for instance by running or jumping away. It remains unclear whether the ‘fearful stimuli’ used in Pavlovian approaches specifically elicits only freezing, or other fear-related behaviors as well.

To investigate this, Chu et al. used high-speed cameras to record rats’ responses to a sound cue they had ‘learned’ to associate with a mild foot shock. In addition to freezing, the animals ran, jumped, stood on their hind legs and stopped their usual reward-seeking behavior in response to the cue. Crucially, these reactions were absent when the rats were exposed to sound cues not associated with pain.

Overall, these experiments demonstrate that Pavlovian conditioning can elicit a full range of fear-related behaviors beyond freezing. Understanding the neural activity behind these diverse responses could lead to more targeted therapies and interventions addressing the various ways stress and anxiety manifest in people.

Animals evolved defensive systems to detect and avoid predation. The predatory imminence continuum (PIC), a prominent theory of defensive behavior, identifies three defensive modes based on the proximity to predation: pre-encounter (leaving the safety of the nest), post-encounter (predator detected), and circa-strike (predation imminent or occurring) ( Fanselow and Lester, 1988 ). Pavlovian fear conditioning has been extensively used to reveal the behavioral and neural underpinnings of defensive systems in rats ( Bolles and Collier, 1976 ; Fanselow, 1993 ; Killcross et al., 1997 ; McNally et al., 2011 ). In a typical Pavlovian fear conditioning procedure, a rat is placed in a neutral context, and played an auditory cue whose termination coincides with foot shock delivery. Each PIC mode is characterized by a unique set of behaviors and, critically, each mode is thought to be captured by a unique epoch of a Pavlovian fear conditioning trial ( Fanselow et al., 2019 ). The post-encounter mode is characterized by freezing, and is captured by cue presentation. Circa-strike is characterized by locomotion, jumping, and rearing, and is captured by shock delivery.

Freezing to a fear conditioned cue may be the most ubiquitous finding in all of behavioral neuroscience ( Blanchard and Blanchard, 1969 ; Bolles and Collier, 1976 ; Maren et al., 1997 ; Anagnostaras, 1999 ; Wilensky et al., 1999 ; Quirk, 2002 ; Koo et al., 2004 ; Rogers and Kesner, 2004 ; Iordanova et al., 2006 ; Shumake et al., 2014 ; Foilb et al., 2016 ; Furlong et al., 2016 ). The relationship between freezing and Pavlovian fear conditioning is so strong that failing to observe freezing in defensive settings has been used to support assertions that Pavlovian fear conditioning did not occur ( Zambetti et al., 2021 ). Cued fear as freezing has been further entrenched by historical observations that locomotion, jumping, and rearing (theorized circa-strike behaviors) are not elicited by fear conditioned cues ( Fanselow et al., 2019 ). Instead, activity-promoting defensive behaviors are restricted to shock delivery ( Fanselow, 1982 ) or to other sudden changes in stimuli ( Fadok et al., 2017 ; Totty et al., 2021 ). Yet, locomotion, jumping, and rearing all readily occur in defensive settings ( Blanchard et al., 1986 ; Holland, 1979 ; Dielenberg and McGregor, 2001 ). Most relevant, a fear conditioned cue can elicit locomotion, rapid forward movements termed ‘darting’ ( Gruene et al., 2015 ; Mitchell et al., 2022 ).

The ability of a fear conditioned cue to elicit locomotion has been called into question ( Trott et al., 2022 ). Trott et al. noted that in prior studies locomotion was greatest at cue onset – the time point most distal from shock delivery ( Gruene et al., 2015 ; Fadok et al., 2017 ). Moreover, prior studies did not use associative controls (but see Totty et al., 2021 ) – essential to making claims that cue-elicited behaviors were due to a predictive relationship with foot shock. Using between-subjects designs in mice, Trott et al. ascribe the majority of cue-elicited locomotion to non-associative cue properties. The foundational study demonstrating the need for proper associative controls in any form of conditioning used Pavlovian fear conditioning ( Rescorla, 1967 ). Not just all-or-none, the magnitude of a fear conditioned, cue-elicited response can scale with foot shock probability ( Rescorla, 1968 ; Ray et al., 2020 ). Rescorla, 1968 many foundational associative learning studies ( Kamin, 1969 ; Rescorla and Wagner, 1972 ), relied on experiments that did not measure ‘fear’ with freezing, but with suppression of operant responding for reward (now termed conditioned suppression) ( Estes and Skinner, 1941 ). Drawing from Rescorla, 1968 , our laboratory has devised a robust, within-subjects Pavlovian fear conditioning procedure in which three cues predict unique foot shock probabilities: danger (p = 1), uncertainty (p = 0.25), and safety (p = 0). Measuring conditioned suppression, we consistently observe complete behavioral discrimination: danger elicits greater suppression than safety, and uncertainty elicits suppression intermediate to danger and safety ( Wright et al., 2015 ; DiLeo et al., 2016 ; Walker et al., 2018 ; Ray et al., 2022 ).

The goal of Experiment 1 was to construct comprehensive, temporal ethograms of rat behavior during discriminative Pavlovian fear conditioning, consisting of a danger, uncertainty, and safety cue. This would allow us to determine what behaviors come under the control of a fear conditioned cue, and how these behaviors are temporally organized. We had the ability to reveal freezing as the exclusive conditioned behavior, as prior studies have found positive relationships between conditioned freezing and conditioned suppression ( Bouton and Bolles, 1980 ; Mast et al., 1982 ). Yet, we also had the ability to detect additional behaviors, as brain manipulations that impair conditioned freezing can have little or no impact on conditioned suppression ( McDannald, 2010 ; McDannald and Galarce, 2011 ). A subgoal was to compare behaviors elicited by the deterministic danger cue, and the probabilistic uncertainty cue. The goal of Experiment 2 was to reveal which of these danger-elicited behaviors transferred to an extinction context in which shock and reward were not present. For Experiment 2, we simplified the discrimination procedure to include only the danger and safety cues.

Twenty-four rats (12 females; Experiment 1) and sixteen rats (8 females, Experiment 2) received Pavlovian fear discrimination. TTL-triggered GigE cameras were installed in behavioral boxes and programmed to capture frames at subsecond temporal resolution prior to and during cue presentation. 86,400 frames (Experiment 1) and 25,600 frames (Experiment 2) were hand scored for nine discrete behaviors reflecting reward ( Holland, 1977 ), activity-suppressing fear ( Blanchard and Blanchard, 1969 ; Fanselow, 1982 ), and activity-promoting fear ( Blanchard et al., 1986 ; Dielenberg and McGregor, 2001 ; Gruene et al., 2015 ). Complete temporal ethograms were constructed during early, middle, and late conditioning sessions (Experiment 1), and for the two types of extinction tests (Experiment 2). Danger responding was compared to baseline and to safety, which served as an unpaired control cue. Behaviors elicited by the danger cue were considered associative (due to pairing with foot shock) if they differed both from baseline and from the safety cue. The temporal profile of responding was determined by tracking behavior change over cue presentation.

Experiment 1

Conditioned suppression reveals complete discrimination.

Twenty-four Long Evans rats (12 females) were trained to nose poke in a central port for food reward. Nose poking was reinforced on a 60-s variable interval schedule throughout behavioral testing. Independent of the poke-food contingency, auditory cues were played through overhead speakers, and foot shock delivered through the grid floor ( Figure 1A ). The experimental design consisted of three cues predicting unique foot shock probabilities: danger (p = 1), uncertainty (p = 0.25), and safety (p = 0) ( Figure 1B ). Behavior chambers were equipped with TTL-triggered cameras capturing 5 frames/s starting 5 s prior to cue presentation and continuing throughout the 10 s cue. TTL-triggered capture yielded 75 frames per trial, and 1200 frames per session. We aimed to capture 28,800 frames each session (1200 frames × 24 rats).

Experimental design and nose poke suppression.

( A ) Conditioned suppression procedure during which rats nose poke for food, while cues are played overhead and shocks delivered through floor. ( B ) Fear discrimination consisted of 10 s auditory cues predicting unique foot shock probabilities: danger (red; p = 1), uncertainty (purple; p = 0.25), and safety (blue; p = 0). Five video frames were captured per second, starting 5 s prior to cue onset and continuing through cue presentation. Mean ± standard error of the mean (SEM) suppression ratios for danger (red), uncertainty (purple), and safety (blue) from pre-exposure through discrimination session 16 are shown for ( C ) females and ( D ) males. Mean + individual suppression ratios for each cue are shown for ( E ) session 2, ( F ) session 8, and ( G ) session 16. Individuals represented by black (female) and gray (male) dots.+95% bootstrap confidence interval does not contain zero.

Our laboratory routinely observes complete behavioral discrimination between danger, uncertainty, and safety in female and male rats measuring conditioned suppression ( Walker et al., 2018 ; Wright et al., 2019 ; Ray et al., 2022 ). Suppression ratios are calculated using baseline and cue nose poke rates: (baseline − cue)/(baseline + cue). Suppression ratios provide a continuous behavior measure, from no suppression (ratio = 0) to total suppression (ratio = 1). To determine if we observed complete behavioral discrimination in these 24 rats, we performed analysis of variance (ANOVA) for suppression ratios [factors: cue (danger vs. uncertainty vs. safety), session (17 total: 1 pre-exposure and 16 discrimination), and sex (female vs. male)]. Complete behavioral discrimination emerged over testing ( Figure 1C, D ). ANOVA found a significant main effect of cue and a significant cue × session interaction ( F s > 6, ps < 0.0001; see Supplementary file 1 for specific values). Sex effects were apparent; ANOVA found a significant main effect of sex, as well as a significant cue × sex interaction and a cue × session × sex interaction ( F s > 3, ps < 0.05; Supplementary file 1 ). Female suppression ratios were higher to each cue across all discrimination sessions: danger ( t 22 = 3.36, p = 0.003), uncertainty ( t 22 = 7.14, p = 3.67 × 10 −7 ), and safety ( t 22 = 4.40, p = 0.0002).

Sex differences in body weight and baseline nose poke rate existed prior to and throughout discrimination, with males weighing more and poking more than females ( Figure 1—figure supplement 1 ). It is therefore possible that sex indirectly moderates conditioned suppression through effects on body weight or baseline nose poke rate. To determine this, we performed analysis of covariance (ANCOVA) for suppression ratios [factors: cue (danger vs. uncertainty vs. safety) and session (17 total: 1 pre-exposure and 16 discrimination)] using body weight or baseline nose poke rate as the covariate. ANCOVA with body weight found neither a significant body weight × cue interaction ( F (2,44) = 2.97, p = 0.062) nor a significant body weight × cue × session interaction ( F (32,704) = 1.40, p = 0.074). However, ANCOVA with baseline nose poke rate found a significant baseline × cue interaction ( F (2,44) = 5.49, p = 0.007) but not a significant baseline × cue × session interaction ( F (32,704) = 0.79, p = 0.79). Irrespective of sex, higher baseline nose poke rates predicted greater discrimination of danger and uncertainty ( Figure 1—figure supplement 2 ).

Constructing behavioral ethograms for all 16 discrimination sessions would have required hand scoring 460,800 frames. To make scoring feasible and capture the emergence of discrimination, we selected sessions 2, 8, and 16. Suppression generalized to all cues during session 2 ( Figure 1E ). Behavioral discrimination emerged by session 8 ( Figure 1F ), and was at its most complete during session 16 ( Figure 1G ). Patterns were confirmed with 95% bootstrap confidence intervals (BCIs) which found no suppression ratio differences for any cue pair during session 2 (all 95% BCIs contained zero), but differences between all cue pairs during sessions 8 and 16 (no 95% BCIs contained zero).

Frames were hand scored for nine discrete behaviors: cup, freezing, grooming, jumping, locomotion, port, rearing, scaling, and stretching, plus ‘background’ (definitions in Supplementary file 2 ). Behavior categories and their definitions were based on prior work in appetitive conditioning ( Holland, 1977 ), foot shock conditioning ( Fanselow, 1982 ; Blanchard et al., 1986 ), as well as our own observations. Representative behavior frames are shown in Figure 2 , Videos 1 – 4 show example danger trials for four different rats (females in Videos 1 and 3 , males in Videos 2 and 4 ).

Representative behaviors.

Representatives frames are shown for: ( A ) background, ( B ) groom, ( C ) port, ( D ) cup, ( E ) locomote, ( F ) jump, ( G ) scale, ( H ) rear, ( I ) stretch, and ( J ) freeze.

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Behavior during a single danger trial.

Video shows the 75 sequential frames for a danger trial. Frames 1–25 are background and 26–75 are danger cue presentation. Observer judgment is shown in the top right for each frame. The specific trial is 23_16_12 (female rat 23, session 16, trial 12).

Temporal ethograms reveal shifting behavioral patterns over discrimination

The 86,400 scored frames allowed us to construct temporal ethograms for danger ( Figure 3A–C ), uncertainty ( Figure 3D–F ), and safety ( Figure 3G–I ) during sessions 2 ( Figure 3 , column 1), 8 ( Figure 3 , column 2), and 16 ( Figure 3 , column 3). Hand scoring showed high inter-rater reliability even when many behaviors were present in a single trial ( Figure 3—figure supplement 1 ). Shifts in the composition of behavior from baseline to cue presentation were apparent across all ethograms. During session 2 (column 1), behavioral shifts lacked cue specificity. Temporal ethograms revealed danger, uncertainty, and safety to equally suppress grooming, port, and cup behavior, but increase freezing, and locomotion. Generalized cue control of behavior was supported by multiple analysis of variance (MANOVA) for all nine behavior categories [factors: cue (danger vs. uncertainty vs. safety), time (15 1 s bins: 5 s baseline → 10 s cue), and sex (female vs. male)] revealing a significant main effect of time ( F (126,2772) = 2.37, p = 5.93 × 10 −15 ), but neither a significant main effect of cue ( F (18,74) = 1.00, p = 0.47) nor a significant cue × time interaction ( F (252,5544) = 1.12, p = 0.11). Cue-specific shifts in behavior were apparent by session 8 (column 2), and continued to session 16 (column 3). Now, MANOVA revealed significant main effects of cue (session 8, F (18,74) = 3.39, p = 0.0001; session 16, F (18,74) = 4.44, p = 0.000002), and significant cue × time interactions (session 8, F (252,5544) = 1.52, p = 3.31 × 10 −8 ; session 16, F (252,5544) = 1.52, p = 4.74 × 10 −7 ). Female-only ethograms are shown in Figure 3—figure supplement 2 ; male-only in Figure 3—figure supplement 3 .

Temporal ethograms.

Mean percent behavior from 5 s prior through 10 s cue presentation is shown for the danger cue during sessions ( A ) 2, ( B ) 8, and ( C ) 16; the uncertainty cue during sessions ( D ) 2, ( E ) 8, and ( F ) 16; and the safety cue during sessions ( G ) 2, ( H ) 8, and ( I ) 16. Behaviors are groom (gray), port (dark purple), cup (light purple), locomote (blue), jump (dark green), scale (light green), rear (yellow), stretch (orange), and freeze (red).

Danger orchestrates a suite of behaviors

A central question driving this study is what behaviors come under the specific control of the fear conditioned, danger cue? To determine this, we focused on session 16, when discrimination was at its most complete. We first performed MANOVA for the 5 s baseline period [factors: cue (danger vs. uncertainty vs. safety), time (5, 1 s bins), and sex (female vs. male)]. As expected, MANOVA returned no main effect of cue, time, nor a cue × time interaction ( F s < 1.5, ps > 0.1). Univariate ANOVA results were subjected to Bonferroni correction (p < 0.0055, 0.05/9 = 0.0055) to account for the nine separate analyses. Like for MANOVA, univariate ANOVA for each of the nine behaviors showed no main effect of cue, time, nor a cue × time interaction. In contrast to all other behaviors, univariate ANOVA for baseline freezing showed a main effect of sex ( F (1,22) = 10.37, p = 0.004). ANOVA for freezing across the baseline and cue periods revealed a significant sex × cue × time interaction ( F (28,616) = 1.94, p = 0.003). Females only froze during early danger presentation while males froze for the duration of danger presentation. The unique freezing pattern warrants separate consideration, which we return to later.

MANOVA was then performed for the 10 s cue period [factors: cue (danger vs. uncertainty vs. safety), time (10, 1 s bins), and sex (female vs. male)]. MANOVA returned significant main effects of cue and time, as well as a significant cue × time interaction ( F s > 1.3, ps < 0.005). Of most interest, univariate ANOVA found a significant main effect of cue for six of the nine behaviors: port ( F (2,44) = 32.15, p = 2.47 × 10 −9 , Figure 4A ), cup ( F (2,44) = 18.40, p = 0.00002, Figure 4B ), locomote ( F (2,44) = 6.33, p = 0.004, Figure 4C ), jump ( F (2,44) = 10.90, p = 0.0001, Figure 4D ), rear ( F (2,44) = 8.64, p = 0.001, Figure 4E ), and freeze ( F (2,44) = 13.86, p = 0.00002). Danger suppressed port and cup behavior ( Figure 4A, B , line graphs), but promoted locomotion, jumping, and rearing ( Figure 4C–E , line graphs). Danger-specific control of behavior was most apparent in the last 5 s of cue presentation ( Figure 4 , shaded region).

Danger-elicited behaviors.

Line graphs show mean ± standard error of the mean (SEM) percent behavior from 5 s prior through 10 s cue presentation for danger (red), uncertainty (purple), and safety (blue) for ( A ) port, ( B ) cup, ( C ) locomote, ( D ) jumping, and ( E ) rearing. Bar plots show mean change in behavior from baseline (5 s prior to cue) compared to last 5 s of cue. Individuals represented by black (female) and gray (male) dots.+95% bootstrap confidence interval for danger vs. safety (black), danger vs. baseline (red), or safety vs. baseline (blue) comparison does not contain zero (black).

Claiming danger specificity requires that % behavior during the danger cue differs from baseline as well as the safety cue. To test this, we subtracted mean % behavior during the 5 s baseline from mean % behavior during the last 5 s of cue presentation, giving %∆ danger, %∆ uncertainty, and %∆ safety for each subject. We constructed 95% BCIs for each cue/behavior. 95% BCIs for %∆ danger did not contain zero for each of the five behaviors ( Figure 4 ), meaning that levels of behavior during cue presentation differed from baseline. Danger presentation decreased port and cup behavior below baseline, but increased locomotion, jumping, and rearing over baseline. 95% BCIs for %∆ uncertainty revealed increased locomotion and jumping, while 95% BCIs for %∆ safety revealed only decreased rearing. To demonstrate danger specificity, we subtracted %∆ safety from %∆ danger. We then constructed 95% BCIs for the difference score for each behavior. Confirming danger specificity (greater changes for danger than for safety), 95% BCIs did not contain zero for each of the five behaviors. Thus, danger specifically and selectively suppressed reward-related port and cup behavior, but promoted locomotion, jumping, and rearing.

Associatively acquired behaviors generalize early

By the end of session 16 each rat had received 96 total foot shocks. It is possible that danger-specific control of multiple behaviors was only observed in session 16 because rats received far more cue–shock pairings than a typical Pavlovian conditioning procedure employs. Session 2 provided a comparison to numbers of cue–shock pairings more typical of fear conditioning studies; rats had received 12 total foot shocks by session’s end. The key question was whether pattern of danger-elicited behaviors in session 2 resembled the pattern in session 16, or if a fundamentally different pattern was observed. To determine this, we performed univariate ANOVA for danger [factors: session (2 vs. 16) and time (15, 1 s bins)] for each of the five behaviors showing session 16 selectivity ( Figure 4—figure supplement 1 ). Confirming near identical temporal patterns of behavior expression during sessions 2 and 16, ANOVA found no significant session × time interaction for any behavior [port ( F (14,322) = 0.45, p = 0.96), cup ( F (14,322) = 0.61, p = 0.86), locomote ( F (14,322) = 1.09, p = 0.37), jump ( F (14,322) = 1.23, p = 0.25), and rear ( F (14,322) = 0.92, p = 0.54)]. Thus, danger orchestrated a suite of behaviors even early in discrimination. Recall that early discrimination (session 2) was marked by non-specific cue control of behaviors. This would mean that associatively acquired behaviors initially generalized to uncertainty and safety – and that discrimination consisted of restricting behavior to danger. In support, univariate ANOVA for session 2 [factors: cue (danger vs. uncertainty vs. safety), time (15, 1 s bins), and sex (female vs. male)] found no cue × time interaction for any of the five, danger-specific behaviors (all F s < 1.2, all ps > 0.3).

Sex informs the temporal pattern of freezing

We return to the case of freezing; the most measured overt fear conditioned behavior. We again focus on session 16 during which discrimination was most complete. Female and male rats differed in the temporal pattern and cue specificity of freezing. Females showed higher baseline freezing levels, a rapid increase in freezing that was specific to danger in the first 5 s, then became non-specific and declined back to baseline levels in the last 5 s ( Figure 5A ). In contrast, males show little baseline freezing and danger-specific freezing increases that persisted throughout cue presentation ( Figure 5B ). Baseline freezing differences were confirmed with independent samples t -test ( t 22 = 3.22, p = 0.0039; Figure 5C ). Confirming sex differences in the temporal pattern of freezing, differential freezing to danger and safety was equivalent in females and males during early cue presentation ( t 22 = 0.02, p = 0.98; Figure 5D , left), but differed during late cue ( t 22 = 2.80, p = 0.01; Figure 5D , right). Generalized freezing to all cues was observed during session 2, with freezing increases more evident in males ( Figure 5—figure supplement 1 ). Thus, discrimination consisted of restricting freezing to danger in males, and selectively freezing to early danger presentation in females.

Special case of freezing.

Line graphs show mean ± standard error of the mean (SEM) percent freezing from 5 s prior through 10 s cue presentation for danger (red), uncertainty (purple), and safety (blue) for ( A ) females and ( B ) males. ( C ) Percent freezing during baseline (5 s prior to cue) is shown for females (black) and males (gray). ( D ) Mean differential freezing to danger and safety is shown for females (black, left) and males (gray, right) during early cue (first 5 s of cue, left) and late cue (last 5 s of cue, right). Mean ± SEM percent freezing change from baseline (5 s prior to cue) compared to last 5 s of danger (red), uncertainty (purple), and safety (blue) for (E) females and (F) males.

Danger-elicited behaviors are independently expressed

Danger suppression of reward-related port and cup behaviors could simply be the byproduct of danger-elicited freezing. Such a relationship has previously been reported ( Bouton and Bolles, 1980 ; Mast et al., 1982 ). To examine the relationship between reward-related behaviors and freezing, in addition to other possible behavior–behavior relationships, we calculated %∆ behavior for early (first 5 s) and late (last 5 s) danger presentation for the six danger-elicited behaviors: cup, port, locomote, jump, rear, and freeze. We constructed 12 × 12 matrices containing the R values ( Figure 6A ) and p values ( Figure 6B ) for the Pearson’s correlation coefficient for each behavior–behavior comparison during the two danger periods. Surprisingly, only one behavior–behavior relationship was observed during the early danger presentation period ( Figure 6A , upper left quadrant). Early rearing and early cup behavior were negatively correlated ( R = −0.43, p = 0.03, but note this would not survive Bonferroni correction). Even more, no behavior–behavior relationships were observed during late danger presentation ( Figure 6A , lower right quadrant). These results suggest the six behaviors are more or less expressed independently of one another.

Behavior–behavior correlations.

( A ) A correlation matrix for the six cue-specific behaviors port (dark purple), cup (light purple), locomote (blue), jump (dark green), rear (yellow), and freeze (red) comparing mean percent behavior during early (first 5 s) and late (last 5 s) cue is shown. Lighter red values indicate positive R values, lighter blue values indicate negative R values. Black indicates R = 0. p values associated with each associated R value are shown in ( B ). Black indicates p values greater than 0.05, while increasingly lighter values indicate lower p values.

Maybe our analysis cannot detect behavior–behavior relationships? To test this, we compared behaviors across the early and late danger periods. Now, the correlation matrix revealed a band of positive R values cutting diagonally across the bottom left quadrant. Five of the 6 behaviors showed positive early–late relationships with themselves: cup ( R = 0.51, p = 0.01), port ( R = 0.87, p = 2.67 × 10 −8 ), locomote ( R = 0.48, p = 0.017), rear ( R = 0.71, p = 7.92 × 10 −5 ), and freeze ( R = 0.48, p = 0.017). In other words, changes in cup behavior evident during early danger presentation persisted to late danger presentation. Jumping was an exception to this trend, as there was no relationship between early and late jumping levels to danger. Overall, danger-elicited behaviors were expressed independently of one another.

Experiment 2

In Experiment 2, we aimed to answer two questions: (1) were the danger-elicited behaviors during discrimination in Experiment 1 dependent on foot shock delivery, and (2) were these behaviors due to the presence of the reward apparatus? To answer this, rats received danger vs. safety discrimination, then were given extinction tests with reward apparatus absent or present. During extinction testing, we captured and hand scored behavior frames before, during, and following cue presentation.

Conditioned suppression reveals complete discrimination during extinction with reward apparatus present

Sixteen Long Evans rats (8 females) were trained to nose poke in a central port for food reward as in Experiment 1. Nose poking was reinforced on a 60-s variable interval schedule throughout behavioral testing. Independent of the poke-food contingency, auditory cues were played through overhead speakers, and foot shock delivered through the grid floor ( Figure 7A ). The experimental design consisted of two cues deterministically predicting foot shock: danger (p = 1) and safety (p = 0) ( Figure 7A ).

( A ) Conditioned suppression procedure during which rats nose poke for food, while danger (red; p = 1) and safety (blue; p = 0) cues are played overhead and shocks delivered through floor. ( B ) Mean ± standard error of the mean (SEM) suppression ratios for danger (red) and safety (blue) from pre-exposure through discrimination session 12 are shown for (left) females and (right) males. ( C ) Rats received one extinction test with reward apparatus absent (left), and another with reward apparatus present (right), counterbalanced. Five video frames were captured per second, starting 5 s prior to cue onset and continuing through 5 s after cue offset. ( D ) Mean + individual suppression ratios for each cue are shown for extinction with reward apparatus present. Individuals represented by black (female) and gray (male) dots. +95% bootstrap confidence interval does not contain zero.

To determine if we observed complete behavioral discrimination, we performed ANOVA for suppression ratios [factors: cue (danger vs. safety), session (13 total: 1 pre-exposure and 12 discrimination), and sex (female vs. male)]. Complete behavioral discrimination emerged over testing ( Figure 7B ). ANOVA found a significant main effect of cue and a significant cue × session interaction ( F s > 8, ps < 0.0001; see Supplementary file 3 for specific values). No significant main effect or interactions with sex were observed. Following the 12 discrimination sessions, each rat received two extinction test sessions. In both test sessions each cue was presented four times. In one test session, the nose poke and food cup were absent while in the other test session the nose poke and food cup were present ( Figure 7C ). Test order was fully counterbalanced. 95% BCIs for differential suppression ratio (danger − safety) during extinction test with the reward present revealed complete discrimination ( Figure 7D ). The 95% BCI did not contain zero [lower bound = 0.24, upper bound = 0.60].

We captured 25,600 total frames (800 frames/test × 16 rats × 2 tests) during extinction testing. Frames were hand scored for nine discrete behaviors: cup, freezing, grooming, jumping, locomotion, port, rearing, scaling, and stretching, plus ‘background’ as in Experiment 1, with the exception that if a trial did not have the reward apparatus present, then food cup and nose poke were not scored.

Danger-elicited locomotion peaks when foot shock would have occurred

The 25,600 scored frames allowed us to construct temporal ethograms for danger ( Figure 8A, B ) and safety ( Figure 8C, D ), during the extinction test with reward apparatus absent ( Figure 8 , column 1), and during the extinction test with the reward apparatus present ( Figure 8 , column 2). Hand scoring showed high inter-rater reliability even when many behaviors were present in a single trial ( Figure 8—figure supplement 1 ). Cue-specific changes in behavior during and following cue presentation were evident. In support, MANOVA [factors: sex (female vs. male), test type (absent vs. present), order (absent first vs. present first), cue (danger vs. safety), and time (20 1 s bins: 5 s baseline → 10 s cue → 5 s post cue)] for the seven behaviors common to both tests [groom, locomote, jump, scale, rear, stretch, and freeze] revealed a significant main effect of time ( F (133,1596) = 2.14, p = 9.44 × 10 −12 ) and a significant cue × time interaction ( F (133,1596) = 1.46, p = 0.001).

Temporal ethograms during extinction.

Mean percent behavior from 5 s prior through 5 s following cue offset is shown for the danger cue during extinction with ( A ) reward apparatus absent and ( B ) reward apparatus present; and the safety cue during extinction with ( C ) reward apparatus absent and ( D ) reward apparatus present. Behaviors are groom (gray), port (dark purple), cup (light purple), locomote (blue), jump (dark green), scale (light green), rear (yellow), stretch (orange), and freeze (red). Note, port and cup are not shown for A and C because the food cup and nose port were absent.

Of the seven behaviors, danger only increased locomotion during both test types ( Figure 9A, B ). In support, univariate ANOVA for locomotion [Bonferroni-corrected p < 0.007 (0.05/7 = 0.007); factors: sex (female vs. male), test type (absent vs. present), order (absent first vs. present first), cue (danger vs. safety), and time (20 1 s bins: 5 s baseline → 10 s cue → 5 s post cue)] found a significant cue × time interaction ( F (19,228) = 3.12, p = 0.000026). Danger-elicited locomotion was most prominent following cue offset, around the time shock would have occurred. 95% BCIs revealed danger-elicited locomotion to exceed baseline and safety cue levels during the 5 s post-cue periods for both the Absent ( Figure 9A ) and Present tests ( Figure 9B ). Additionally, the 95% BCI revealed danger-elicited locomotion to exceed safety-elicited locomotion during the late cue period during the Present test, though danger-elicited locomotion did not exceed baseline ( Figure 9B ). Locomotion never increased during safety trials (all 95% BCIs contain zero). Danger-elicited locomotion occurred regardless of test order, as ANOVA revealed no significant order interactions ( F s < 1.5, ps > 0.2). Sex partially mediated the temporal expression of locomotion, with ANOVA finding a significant sex × cue × time interaction ( F (19,228) = 2.34, p = 0.002). Females showed more robust post-cue, danger locomotion during both test types ( Figure 9—figure supplement 1 ). Males showed more robust danger-elicited locomotion during the late cue period during the Present test ( Figure 9—figure supplement 2 ). The results reveal that danger-elicited locomotion transfers to extinction settings when both foot shock and the reward apparatus were absent.

Danger elicits locomotion.

Line graphs show mean ± standard error of the mean (SEM) percent behavior from 5 s prior through 10 s cue presentation for danger (red) and safety (blue) for locomotion during the ( A ) reward apparatus absent extinction test and ( B ) reward apparatus present extinction test. Bar plots show mean change in behavior from baseline (5 s prior to cue) compared to early (first 5 s), late (last 5 s), and post (5 s after offset) cue periods. Individuals represented by black (female) and gray (male) dots. The same is shown for freezing ( C, D ) .+95% bootstrap confidence interval for danger vs. safety (black), danger vs. baseline (red), or safety vs. baseline (blue) comparison does not contain zero (black).

Freezing is less dangerspecific and is sensitive to time, test type, and order

Unlike locomotion, there was lesser evidence of danger-specific freezing during extinction testing ( Figure 9C, D ). Most notably, univariate ANOVA [correction and factors identical to locomotion] found that the cue × time interaction failed to achieve significance ( F (19,228) = 1.25, p = 0.011). When organizing % freezing by test type, there was no period (early cue, late cue, and post cue) during which freezing increases over baseline were selective to danger ( Figure 9C, D ). The only period during which freezing to danger exceeded freezing to safety was the early cue period when the reward apparatus was present ( Figure 9D , right). Though even during this period increases in freezing to safety were observed. Instead, freezing tended to generalize to safety; meaning it was cue evoked but not cue specific. Additionally, freezing was more prominent during extinction testing with the reward apparatus absent. In support, univariate ANOVA revealed significant main effects of time ( F (19,228) = 5.13, p = 3.64 × 10 −10 ) and test ( F (1,12) = 21.20, p = 0.001). Like freezing, neither rearing nor jumping showed evidence of danger specificity with univariate ANOVA for each finding no significant cue × time interaction ( F s < 1.5, ps > 0.2).

However, order mediated the specificity of danger-elicited freezing. Rats receiving the Present test first showed selective and differential freezing to danger ( Figure 9 , Figure 9—figure supplement 3A ). Rats receiving the Absent test first showed no evidence of selective and differential freezing to danger ( Figure 9 , Figure 9—figure supplement 3B ). In support, univariate ANOVA returned a significant order × cue × time interaction ( F (19,228) = 2.14, p = 0.002). Of note, no significant order × cue × time interaction was observed for locomotion ( F (19,228) = 1.03, p = 0.43). The same rats that showed robust danger-elicited locomotion across both test types showed danger-elicited freezing that was sensitive to test order.

Danger-elicited behaviors are independently expressed during extinction

We were interested to see if there were relationships between danger-elicited locomotion and freezing during extinction testing. To determine this we calculated Pearson’s correlation coefficients ( R value) for individual freezing and locomotion (% behavior over baseline) during early, late, and post-danger cue periods in extinction sessions with reward apparatus absent ( Figure 10A, B ) and with reward apparatus present ( Figure 10C, D ). As in Experiment 1, we found no evidence of inhibitory relationships between locomotion and freezing. That is, no comparison found a negative R value. This was true both within and between trial periods. Instead, and like for Experiment 1, correlational analyses reveal significant, positive relationships within behaviors across trial periods. These positive relationships were more prominent during extinction testing with the reward apparatus present. In particular, freezing was positively correlated across all trial periods during the present extinction sessions [early–late R = 0.82, p = 1.05 × 10 −4 ; early–post R = 0.60, p = 0.015; post–late R = 0.68, p = 0.0036]. These results demonstrate that opposing danger-elicited behaviors are independently expressed during extinction.

Behavior–behavior correlations during extinction.

( A ) A correlation matrix for locomote (blue) and freeze (red) comparing mean percent behavior during early (first 5 s), late (last 5 s), and post (5 s after) cue period is shown for the reward absent extinction test. Lighter red values indicate positive R values, lighter blue values indicate negative R values. Black indicates R = 0. p values associated with each associated R value are shown in ( B ). Black indicates p values greater than 0.05, while increasingly lighter values indicate lower p values. Same shown for behavior correlations during reward present extinction test ( C, D ).

We set out to quantify behaviors elicited by a fear conditioned, danger cue. Consistent with virtually all studies of Pavlovian fear conditioning (but see Amorapanth et al., 1999 ), we observed danger-elicited freezing over the course of acquisition (Experiment 1) and during extinction testing (Experiment 2). Yet, freezing was not the dominant danger-elicited behavior. Instead, danger orchestrated a suite of behaviors. During Experiment 1, danger suppressed reward behavior directed toward the site of food delivery and the location of the rewarded action. Even more, danger-elicited locomotion, jumping, and rearing. During Experiment 2, danger again suppressed rewarded action and continued to elicit locomotion. During both experiments, freezing was most prominent at danger cue onset. Locomotion was most prominent toward danger cue offset (Experiment 1), and when shock would have occurred in extinction (Experiment 2).

Before discussing our results further, an important limitation should be raised. 40–50% of frames could not be assigned to a specific behavior and were labeled as background. This was due to three main factors. First, in order to objectively hand score many behaviors, we developed mutually exclusive, specific definitions. Our strict definitions meant erring on the side of labeling a behavior background if there was uncertainty in judgment. Second, use of a single, side view camera meant the observer could not view a rat’s forelimbs or face when the rat was turned away from the camera. If forelimb and face position could not be determined the frame was labeled background. Finally, transition behaviors (e.g., switching from rearing to locomotion) and other behaviors (e.g., sniffing and turning) that did not fit into one of the nine behavior definitions were labeled background. The upside of this limitation is high confidence in behavior judgments and high inter-rater reliability for those judgments.

Studies assessing auditory fear conditioning in a neutral context routinely report freezing to account for >80% of behavior during cue presentation ( Bolles and Collier, 1976 ; Maren et al., 1997 ; Anagnostaras, 1999 ; Wilensky et al., 1999 ; Quirk, 2002 ; Koo et al., 2004 ; Rogers and Kesner, 2004 ; Iordanova et al., 2006 ; Shumake et al., 2014 ; Foilb et al., 2016 ; Furlong et al., 2016 ). The sheer number of demonstrations, and number of groups independently observing dominant freezing, puts us firmly in the minority. Placing us further in the minority, we observe danger-elicited locomotion, jumping, and rearing. These behaviors are characterized by lateral and vertical movements, polar opposites to the immobility that characterizes freezing. A commonality of the studies above was use of contexts in which only cues and shocks were delivered, with shocks being more intense than shocks used in our studies. These experimental settings favor freezing. It is likely that our inclusion of competing, reward behaviors and use of lower shock intensities permitted a broader range of danger-elicited behaviors to be observed ( Holland, 1979 ; Mitchell et al., 2022 ).

Indeed, we are not the first group to observe locomotion, jumping, or rearing in defensive settings in rats. Using more traditional Pavlovian fear conditioning designs, Shansky and colleagues have observed ‘darting’, rapid forward movements across the test chamber, to a fear conditioned cue ( Gruene et al., 2015 ). While more readily observed in female rats, darting can be observed in males under certain experimental conditions ( Mitchell et al., 2022 ). Our definition of locomotion aligns well with the Shansky lab definition of darting. We found that danger-elicited locomotion was equally apparent during extinction, and more robust than freezing. While locomotion was observed across all rats, female locomotion was better timed to shock delivery. Our results join previous studies in demonstrating a fear conditioned cue promotes movement in rats ( Bolles and Collier, 1976 ; Totty et al., 2021 ).

Jumping is elicited in rats by hypoxia (decreasing oxygen levels in the air) – a life-threatening condition ( Spiacci et al., 2015 ). More relevant to our study, the Blanchards observed jumping in defensive settings in rats ( Blanchard et al., 1986 ). In their procedure, a rat was placed at the end of an inescapable hallway, then a human experimenter slowly approached. Rats initially froze when the experimenter was distant (1–2 m away), but switched from freezing to jumping as the experimenter drew near (<1 m). Our observation of danger-elicited jumping during fear acquisition, and its preferential expression at the end of danger presentation, mirrored the defensive jumping pattern observed in the Blanchard’s study.

Rearing ( Lever et al., 2006 ) is elicited by visual cues predicting moderate foot shock. Holland, 1979 found that a mix of rearing and freezing are acquired to a visual cue paired with low intensity foot shock. A visual cue paired with intense foot shock exclusively produces freezing. The foot shock intensity we used in both experiments (0.5 mA) is more consistent with the low intensities in the Holland, 1979 experiment. Dielenberg and McGregor, 2001 found that rats exposed to a recently worn cat collar, with an opposing box to hide in, show ‘vigilant rearing’ to the cat collar ( Dielenberg and McGregor, 2001 ). Rearing was never observed in a control condition. While we cannot claim vigilance, we find that danger promotes rearing during fear acquisition.

Temporal ethograms revealed that during fear acquisition, jumping and rearing were most prevalent at the end of cue presentation – when foot shock was imminent. This was in contrast to freezing which was prominent during early danger presentation for both females and males, but only shown by males at the end of cue presentation. Though unlike Experiment 1, in which foot shock was present, we found no evidence of danger-elicited jumping and rearing during fear extinction. Because jumping and rearing are vertical behaviors, they may be avoidant or escape responses. The rat is trying to leave the floor before the shock comes on. This interpretation is supported by the finding that these responses peaked just before shock presentation in Experiment 1. Removing foot shock in Experiment 2 may have removed the impetus for avoidance and escape. However, it could be equally likely that these behaviors are more sensitive context change. Experiment 2 also found that freezing transferred less well to the extinction context in which reward was absent.

Our findings accord well with the PIC theory of defensive behavior ( Fanselow and Lester, 1988 ). PIC theory identifies three defensive modes: pre-encounter (e.g., leaving the safety of the nest to forage), post-encounter (predator detected), and circa-strike (predation inevitable or occurring). Analogs to PIC modes are identified in a Pavlovian fear conditioning trial ( Fanselow et al., 2019 ). Pre-encounter mode may correspond to leaving the home cage and being placed in the experimental chamber where foot shocks occur. Post-encounter mode corresponds to presentation of the fear conditioned cue. Circa-strike mode is said to correspond to foot shock delivery. It is argued that circa-strike behaviors (locomotion, jumping, and rearing) are not observed toward the end of danger presentation because rats do not time shock delivery. In support, extending cue duration in traditional cued fear conditioning paradigms does not result in shifts from freezing to locomotion, jumping, and rearing toward cue offset ( Fanselow et al., 2019 ).

Here, we find expected patterns of defensive behavior in unexpected epochs of Pavlovian conditioning trials. Early danger freezing by all rats (females and males) gives way to a late mix of danger-elicited behaviors that included locomotion, jumping, and rearing (Experiment 1) or late locomotion (Experiment 2). Why do we observe late danger control of circa-strike behaviors? Hunger and the availability of a rewarded action may provide an impetus for shock timing. Timing would allow rats to minimize the display of defensive behaviors and maximize reward seeking. In support, presenting long duration danger cues in a conditioned suppression setting results in timing of fear responding. With experience, rats show little suppression of reward seeking to danger onset, which ramps toward shock delivery ( Rosas and Alonso, 1996 ). Supporting the minimization of defensive behavior in reward settings, foot shocks signaled by danger will strongly suppress reward seeking only early in fear conditioning. Shock-induced suppression quickly wanes and with experience, shock delivery will paradoxically facilitate reward seeking ( LaBarbera and Caul, 1976 ; Strickland et al., 2021 ). Shock timing information is readily apparent in the ventrolateral periaqueductal gray, a brain region central to defensive behavior ( Fanselow, 1993 ; Carrive et al., 1997 ; Mobbs et al., 2007 ; McDannald, 2010 ; Tovote et al., 2016 ; Arico et al., 2017 ). Populations of ventrolateral periaqueductal gray neurons show little responding upon danger presentation, but ramp firing toward shock delivery ( Ozawa et al., 2017 ; Wright and McDannald, 2019 ; Wright et al., 2019 ). Our results support the PIC theory of defensive behavior but demonstrate that the relationship between defensive mode and Pavlovian conditioning trial epoch is not fixed, but depends on the experimental setting.

A secondary goal of Experiment 1 was to compare defensive behaviors elicited by a deterministic, danger cue and a probabilistic, uncertainty cue. In our setting, uncertainty is not simply a diminished version of danger. Indeed, uncertainty only promoted a subset of danger-elicited behaviors: locomotion and jumping. Most surprising was the inability of uncertainty to suppress reward behaviors directed toward the food cup and port. This is particularly puzzling because using suppression ratios, we found uncertainty to produce robust suppression of nose poking. What is going on here? Food cup, port, and poke behavior lie on a rewarded action continuum. Food cup means the rat is in the area of food delivery – but is most distal from the rewarded action. Port means the rat is around or in the site of the rewarded action, but only poke requires the rat to be fully engaged in performing the rewarded action (nose all the way into the port). Danger suppresses all reward behavior regardless of proximity to rewarded action. In contrast, uncertainty selectively suppresses reward behavior most proximal to the rewarded action.

By comprehensively quantifying behavior and constructing temporal ethograms, we found a fear conditioned cue to independently control at least six distinct behaviors during fear acquisition and three distinct behaviors during fear extinction. Though our study was exclusively behavioral, we feel our results have implications for investigations into the neural basis of fear learning and the organization of defensive behavior. Most important is that a fear conditioned cue does not simply elicit freezing. Behaviors elicited by a fear conditioned cue are the product of many factors: species, sex, age, behavioral setting, and experimenter-determined parameters (CS/US type, duration, and intensity; trial number, inter-trial interval [ITI], and more). In our view, freezing is a common – not dominant – defensive behavior because the field has favored behavioral settings and experimenter-determined parameters that maximize the expression of ‘fear’ through freezing ( McDannald, 2023 ). Here, we show that a relatively simple modification of the rat’s behavioral setting – access to a rewarded action – is sufficient to de-emphasize freezing and promote the expression of many additional behaviors. Most prominent of these is locomotion. Even more, Pavlovian fear conditioning over a baseline of reward seeking reveals a temporally organized sequence of cue-elicited defensive behaviors predicted by PIC theory. The independent expression of these behaviors is appealing for studies attempting to link discrete behavioral sequelae of ‘fear’ to distinct neural circuits, breathing new life into a classic Pavlovian fear conditioning procedure ( Estes and Skinner, 1941 ).

All procedures were performed in the Boston College Animal Care Facility in accordance with NIH and Boston College guidelines. The Boston College experimental protocol supporting these procedures is 2024-001.

For Experiment 1, 24 adult Long Evans rats (12 female) weighing 196–298 g arrived from Charles River Laboratories on postnatal day 55. Rats were single-housed on a 12-hr light cycle (lights off at 6:00 pm) and maintained at their initial body weight with standard laboratory chow (18% Protein Rodent Diet #2018, Harlan Teklad Global Diets, Madison, WI). Water was available ad libitum in the home cage. For Experiment 2, sixteen adult Long Evans rats (eight females) were housed and maintained as described above. All protocols were approved by the Boston College Animal Care and Use Committee and all experiments were carried out in accordance with the NIH guidelines regarding the care and use of rats for experimental procedures.

Behavior apparatus

The apparatus for Pavlovian fear discrimination consisted of four individual chambers with aluminum front and back walls, clear acrylic sides and top, and a grid floor. LED strips emitting 940 nm light were affixed to the acrylic top to illuminate the behavioral chamber for frame capture. 940-nm illumination was chosen because rats do not detect light wavelengths exceeding 930 nm ( Nikbakht and Diamond, 2021 ). Each grid floor bar was electrically connected to an aversive shock generator (Med Associates, St. Albans, VT). An external food cup, and a central port equipped with infrared photocells were present on one wall. Auditory stimuli were generated with an Arduino-based device and presented through two speakers mounted on the ceiling.

Pellet exposure and nose poke shaping

Rats were food restricted and specifically fed to maintain their body weight throughout behavioral testing. Each rat was given 4 g of experimental pellets in their home cage in order to overcome neophobia. Next, the central port was removed from the experimental chamber, and rats received a 30-min session in which one pellet was delivered every minute. The central port was returned to the experimental chamber for the remainder of behavioral testing. Each rat was then shaped to nose poke in the central port for experimental pellet delivery using a fixed ratio schedule in which one nose poke into the port yielded one pellet. Shaping sessions lasted 30 min or until approximately 50 nose pokes were completed. Each rat then received six sessions during which nose pokes into the port were reinforced on a variable interval schedule. Session 1 used a variable interval 30-s schedule (poking into the port was reinforced every 30 s on average). All remaining sessions used a variable interval 60-s schedule. For the remainder of behavioral testing, nose pokes were reinforced on a variable interval 60-s schedule independent of cue and shock presentation.

Cue pre-exposure

Each rat was pre-exposed to the three cues to be used in Pavlovian discrimination in one session. Auditory cues consisted of repeating motifs of broadband click, phaser, or trumpet. This 37-min session consisted of four presentations of each cue (12 total presentations) with a mean ITI of 2.5 min. Trial type order was randomly determined by the behavioral program and differed for each rat, each session.

Pavlovian fear discrimination

Each rat received 16, 48-min sessions of fear discrimination. Each session consisted of 16 trials, with a mean ITI of 2.5 min. Auditory cues were 10 s in duration. Each cue was associated with a unique foot shock probability (0.5 mA, 0.5 s): danger, p = 1.00; uncertainty, p = 0.25; and safety, p = 0.00. Foot shock was administered 2 s following the termination of the auditory cue on danger and uncertainty-shock trials. Auditory identity was counterbalanced across rats. Each session consisted of four danger trials, two uncertainty-shock trials, six uncertainty-omission trials, and four safety trials. Trial type order was randomly determined by the behavioral program and differed for each rat, each session.

Each rat received 12, 48-min sessions of fear discrimination. Each session consisted of eight trials, with a mean ITI of 3.5 min. Auditory cues were 10 s in duration. Each cue was associated with a unique foot shock probability (0.5 mA, 0.5 s): danger, p = 1.00 and safety, p = 0.00. Foot shock was administered 2 s following the termination of the auditory cue on danger trials. Auditory identity was counterbalanced across rats. Each session consisted of four danger trials and four safety trials. Trial type order was randomly determined by the behavioral program and differed for each rat, each session.

Fear extinction

For Experiment 2, each rat received two types of extinction test: one with the port and food cup present and one with the port and food cup absent. Test type order was counterbalanced across rats with half receiving the port and cup present first. Extinction sessions were 48 min in duration, and consisted of four danger and four safety trials, with a mean ITI of 3.5 min. Auditory cues were 10 s in duration. Foot shocks were not delivered. Auditory identity of danger and safety were maintained from discrimination, which was counterbalanced. Trial type order was randomly determined by the behavioral program and differed for each rat.

Calculating suppression ratio

Time stamps for cue presentations, shock delivery, and nose pokes (photobeam break) were automatically recorded by the Med Associates program. Baseline nose poke rate was calculated for each trial by counting the number of pokes during the 20 s pre-cue period and multiplying by 3. Cue nose poke rate was calculated for each trial by counting the number of pokes during the 10 s cue period and multiplying by 6. Nose poke suppression was calculated as a ratio: (baseline poke rate − cue poke rate)/(baseline poke rate + cue poke rate). A suppression ratio of ‘1’ indicated complete suppression of nose poking during cue presentation relative to baseline. A suppression ratio of indicated ‘0’ indicates equivalent nose poke rates during baseline and cue presentation. Gradations in suppression ratio between 1 and 0 indicated intermediate levels of nose poke suppression during cue presentation relative to baseline. Negative suppression ratios indicated increased nose poke rates during cue presentation relative to baseline.

Frame capture system

Behavior frames were captured using Imaging Source monochrome cameras (DMK 37BUX28; USB 3.1, 1/2.9″ Sony Pregius IMX287, global shutter, resolution 720 × 540, trigger in, digital out, C/CS-mount). Frame capture was triggered by the Med Associates behavior program. The 28 V Med Associates pulse was converted to a 5-V TTL pulse via Adapter (SG-231, Med Associates, St. Albans, VT). The TTL adapter was wired to the camera’s trigger input. Captured frames were saved to a PC (OptiPlex 7470 All-in-One) running IC Capture software (Imaging Source). For Experiment 1, frame capture began precisely 5 s before cue onset and continued throughout 10 s cue presentation. Frames were captured at a rate of 5 per second, with a target of capturing 75 frames per trial (5 frames/s × 15 s = 75 frames), and 1200 frames per session (75 frames/trial × 16 trials = 1200 frames). For Experiment 2, frame capture began 5 s before cue onset and continued throughout 10 s cue presentation and 5 s after cue termination. Frames were captured at a rate of 5 per second, with a target of capturing 100 frames per trial (5 frame/s × 20 s = 100 frames), and 800 frames per session (100 frames/trial × 8 trials = 800 frames).

Post-acquisition frame processing

We aimed to capture 1200 frames per session, and selected sessions 2, 8, and 16 for hand scoring. A Matlab script sorted the 1200 frames into 16 folders, one for each trial, each containing 75 frames. Each 75-frame trial was made into a 75-slide PowerPoint presentation to be used for hand scoring.

We aimed to capture 800 frames per session, and selected extinction sessions 1 and 2 for hand scoring. A Matlab script sorted the 800 frames into 8 folders, one for each trial, each containing 100 frames. Each 100-frame trial was made into a 100-slide PowerPoint presentation to be used for hand scoring.

Anonymizing trial information

For Experiment 1, a total of 1152 trials of behavior were scored from the 24 rats over the 3 sessions of discrimination (16 trials per session). For Experiment 2, a total of 256 trials were scored from 16 rats over the 2 extinction sessions (8 trials per session). We anonymized trial information in order to score behavior without bias. The numerical information from each trial (session #, rat #, and trial #) was encrypted as a unique number sequence. A unique word was then added to the front of this sequence. The result was that each of the trials was converted into a unique word + number sequence. For example, trial ac01_02_07 (rat #1, session #2, and trial #7) would be encrypted as: abundant28515581. The trials from Experiment 1 were randomly assigned to five observers. 256 trials from Experiment 2 were randomly assigned to seven observers. The result of trial anonymization was that observers were completely blind to subject, trial type, and session number. Furthermore, random assignment meant that the 16 or 8 trials composing a single session were scored by different observers.

Behavior categories and definitions

Frames were scored as one of ten mutually exclusive behavior categories, defined as follows:

Specific behavior cannot be discerned because the rat is turned away from the camera or position of forepaws is not clear, or because the rat is not engaged in any of the other behaviors.

Any part of the nose above the food cup but below the nose port.

Arched back and stiff, rigid posture in the absence of movement, all four limbs on the floor (often accompanied by hyperventilation and piloerection). Side-to-side head movements and up and down head movements that do not disturb rigid posture are permitted. Activity such as sniffing or investigation of the bars is not freezing. Freezing, as opposed to pausing, is likely to be 3 or more frames (600+ms) long.

Any scratching, licking, or washing of the body.

All four limbs off the floor. Includes hanging which is distinguished when hind legs are hanging freely.

Propelling body across chamber on all four feet, as defined by movement of back feet. Movement of back feet with front feet off the floor is rearing.

Any part of the nose in the port. Often standing still in front of the port but sometimes tilting head sideways with the body off to the side of the port.

One or two hind legs on the grid floor with both forepaws o ff the grid floor and not on the food cup. Usually (not always) stretching to full extent, forepaws usually (not always) on top of side walls of the chamber, often pawing walls; may be accompanied by sniffing or slow side-to-side movement of head. Does not include grooming movements or eating, even if performed while standing on hind legs.

All four limbs off the floor but at least two limbs on the side of the chamber. Standing on the food cup counts as scaling.

Body is elongated with the back posture ‘flatter’ than normal. Stretching is often accompanied by immobility, like freezing, but is distinguished by the shape of the back.

Frame scoring system

Frames were scored using a specific procedure. Frames were first watched in real time in Microsoft PowerPoint by setting the slide duration and transition to 0.19 s, then playing as a slideshow. Behaviors clearly observed were noted. Next, the observer went through all the frames scoring one behavior at a time. A standard scoring sequence was used: port, cup, rear, scale, jump, groom, freeze, locomote, and stretch. When the specific behavior was observed in a frame, that frame was labeled. Once all behaviors had been scored, the video was re-watched for freezing. The unlabeled frames were then labeled ‘background’. Finally, all background frames were checked to ensure they did not contain a defined behavior.

Inter-observer reliability

To assess inter-observer reliability, we selected 12 trials from outside sessions 2, 8, and 16, six from females and six from males. Each of our five observers scored these 12 trials, interweaving the 12 comparison trials with the primary data trials. As a result, each observer scored 900 comparison frames which were then used to assess inter-observer reliability.

Inter-observer reliability was assessed as described in Experiment 1. Eight trials from outside extinction sessions 1 and 2 were selected for comparison. Each observer scored 800 comparison frames which were then used to assess inter-observer reliability.

Statistical analyses

ANOVA was performed for body weight, baseline nose poke rate, suppression ratios, and specific behaviors. Sex was used as a factor for all analyses. Cue, session, and time were used as factors when relevant. Univariate ANOVA following MANOVA used a Bonferroni-corrected p value significance of 0.0055 (0.05/9) to account for the nine quantified behaviors. MANOVA was performed for the nine quantified behaviors with factors of sex, cue, and time. Pearson’s correlation coefficient was used to examine the relationship between baseline nose poke rate and body weight, baseline nose poke rate and cue discrimination, as well as the relationship between danger cue-elicited behaviors during early and late cue presentation in session. Within-subject comparisons were made using 95% BCIs with the Matlab bootci function. Comparisons were said to differ when the 95% BCI did not contain zero. Between subject’s comparisons were made using independent samples t -test.

Raw images and observer judgments are freely available: https://doi.org/10.7910/DVN/HKMUUN .

Hanrahan KE
Williams DC
McDannald MA
Amorapanth P
Google Scholar
Anagnostaras S
Badrinarayan A
Vander Weele CM
Saunders BT
Couturier BE
Blanchard RJ
Blanchard DC
Flannelly KJ
Dielenberg RA
McGregor IS
Kovacevic A
Fanselow MS
Flyer-Adams JG
Christianson JP
Richardson R
Iordanova MD
Westbrook RF
Killcross AS
Killcross S
LaBarbera JD
Johansen JP
Mitchell JR
Wasielewski S
Huckleberry KA
Marchant JL
Rescorla RA
Furgeson-Moreira S
de O Sergio T
da Silva GSF
Schenberg LC
Garcia-Cairasco N
Zangrossi H
Strickland JA
Huddleston I
Ramanathan KR
Oleksiak CR
Esposito MS
Ramakrishnan C
Deisseroth K
Andreansky C
Wilensky AE
Pimpinelli D
Zambetti PR
Schuessler BP

Author details

Contribution, for correspondence, competing interests, additional information.

National Institutes of Health (MH117791)

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

All protocols were approved by the Boston College Animal Care and Use Committee and all experiments were carried out in accordance with the NIH guidelines regarding the care and use of rats for experimental procedures. The Boston College experimental protocol supporting these procedures is 2024-001.

This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited.

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Cite this article (links to download the citations from this article in formats compatible with various reference manager tools), categories and tags, research organism, further reading, lost in translation: inconvenient truths on the utility of mouse models in alzheimer’s disease research.

The recent, controversial approval of antibody-based treatments for Alzheimer’s disease (AD) is fueling a heated debate on the molecular determinants of this condition. The discussion should also incorporate a critical revision of the limitations of preclinical mouse models in advancing our understanding of AD. We critically discuss the limitations of animal models, stressing the need for careful consideration of how experiments are designed and results interpreted. We identify the shortcomings of AD models to recapitulate the complexity of the human disease. We dissect these issues at the quantitative, qualitative, temporal, and context-dependent levels. We argue that these models are based on the oversimplistic assumptions proposed by the amyloid cascade hypothesis (ACH) of AD and fail to account for the multifactorial nature of the condition. By shedding light on the constraints of current experimental tools, this review aims to foster the development and implementation of more clinically relevant tools. While we do not rule out a role for preclinical models, we call for alternative approaches to be explored and, most importantly, for a re-evaluation of the ACH.

Task-dependent coarticulation of movement sequences

Combining individual actions into sequences is a hallmark of everyday activities. Classical theories propose that the motor system forms a single specification of the sequence as a whole, leading to the coarticulation of the different elements. In contrast, recent neural recordings challenge this idea and suggest independent execution of each element specified separately. Here, we show that separate or coarticulated sequences can result from the same task-dependent controller, without implying different representations in the brain. Simulations show that planning for multiple reaches simultaneously allows separate or coarticulated sequences depending on instructions about intermediate goals. Human experiments in a two-reach sequence task validated this model. Furthermore, in co-articulated sequences, the second goal influenced long-latency stretch responses to external loads applied during the first reach, demonstrating the involvement of the sensorimotor network supporting fast feedback control. Overall, our study establishes a computational framework for sequence production that highlights the importance of feedback control in this essential motor skill.

High-frequency terahertz stimulation alleviates neuropathic pain by inhibiting the pyramidal neuron activity in the anterior cingulate cortex of mice

Neuropathic pain (NP) is caused by a lesion or disease of the somatosensory system and is characterized by abnormal hypersensitivity to stimuli and nociceptive responses to non-noxious stimuli, affecting approximately 7–10% of the general population. However, current first-line drugs like non-steroidal anti-inflammatory agents and opioids have limitations, including dose-limiting side effects, dependence, and tolerability issues. Therefore, developing new interventions for the management of NP is urgent. In this study, we discovered that the high-frequency terahertz stimulation (HFTS) at approximately 36 THz effectively alleviates NP symptoms in mice with spared nerve injury. Computational simulation suggests that the frequency resonates with the carbonyl group in the filter region of Kv1.2 channels, facilitating the translocation of potassium ions. In vivo and in vitro results demonstrate that HFTS reduces the excitability of pyramidal neurons in the anterior cingulate cortex likely through enhancing the voltage-gated K + and also the leak K + conductance. This research presents a novel optical intervention strategy with terahertz waves for the treatment of NP and holds promising applications in other nervous system diseases.

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Little Albert Experiment (Watson & Rayner)

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Watson and Rayner (1920) conducted the Little Albert Experiment to answer 3 questions:

Can an infant be conditioned to fear an animal that appears simultaneously with a loud, fear-arousing sound?

Would such fear transfer to other animals or inanimate objects?

How long would such fears persist?

Little Albert Experiment

Ivan Pavlov showed that classical conditioning applied to animals. Did it also apply to humans? In a famous (though ethically dubious) experiment, John B. Watson and Rosalie Rayner showed it did.

Conducted at Johns Hopkins University between 1919 and 1920, the Little Albert experiment aimed to provide experimental evidence for classical conditioning of emotional responses in infants

At the study’s outset, Watson and Rayner encountered a nine-month-old boy named “Little Albert” (his real name was Albert Barger) – a remarkably fearless child, scared only by loud noises.

After gaining permission from Albert’s mother, the researchers decided to test the process of classical conditioning on a human subject – by inducing a further phobia in the child.

The baseline session occurred when Albert was approximately nine months old to test his reactions to neutral stimuli.

Albert was reportedly unafraid of any of the stimuli he was shown, which consisted of “a white rat, a rabbit, a dog, a monkey, with [sic] masks with and without hair, cotton wool, burning newspapers, etc.” (Watson & Rayner, 1920, p. 2).

Approximately two months after the baseline session, Albert was subjected during two conditioning sessions spaced one week apart to a total of seven pairings of a white rat followed by the startling sound of a steel bar being struck with a hammer.

When Little Albert was just over 11 months old, the white rat was presented, and seconds later, the hammer was struck against the steel bar.

After seven pairings of the rat and noise (in two sessions, one week apart), Albert reacted with crying and avoidance when the rat was presented without the loud noise.

By the end of the second conditioning session, when Albert was shown the rat, he reportedly cried and “began to crawl away so rapidly that he was caught with difficulty before reaching the edge of the table” (p. 5). Watson and Rayner interpreted these reactions as evidence of fear conditioning.

By now, little Albert only had to see the rat and immediately showed every sign of fear. He would cry (whether or not the hammer was hit against the steel bar), and he would attempt to crawl away.

The two conditioning sessions were followed by three transfer sessions. During the first transfer session, Albert was shown the rat to assess maintained fear, as well as other furry objects to test generalization.

Complicating the experiment, however, the second transfer session also included two additional conditioning trials with the rat to “freshen up the reaction” (Watson & Rayner, 1920, p. 9), as well as conditioning trials in which a dog and a rabbit were, for the first time, also paired with the loud noise.

This fear began to fade as time went on, however, the association could be renewed by repeating the original procedure a few times.

Unlike prior weekly sessions, the final transfer session occurred after a month to test maintained fear. Immediately following the session, Albert and his mother left the hospital, preventing Watson and Rayner from carrying out their original intention of deconditioning the fear they have classically conditioned.

Experimental Procedure

Session	Age	Stimuli Shown
	8 months & 26 days	Included tests with rat, rabbit, dog, monkey, masks with and without hair, cotton wool, and burning newspapers (no fear).
	11 months & 3 days	Rat paired with loud noise (two pairings).
	11 months & 10 days	Test with rat alone (elicited mild fear). Rat paired with loud noise (5 pairings). Test with rat alone (elicited strong fear).
	11 months & 15 days	Tests with rat, rabbit, dog, fur coat, cotton wool, Watson’s hair, 2 observers’ hair, and Santa Claus mask.
	11 months & 20 days	In original testing room: tests with rat, rabbit, and dog; an extra conditioning trial with rat; and conditioning trials with rabbit and dog (1 pairing each). In a new room: tests with rat, rabbit, and dog; extra conditioning trial with rat; plus barking incident with dog. Included comment that all previous tests had been conducted on a table.
	12 months, 21 days	Tests with Santa Claus mask, fur coat, rat, rabbit, and dog. Albert was also discharged from the hospital on this day.

Classical Conditioning

Neutral Stimulus (NS): This is a stimulus that, before conditioning, does not naturally bring about the response of interest. In this case, the Neutral Stimulus was the white laboratory rat. Initially, Little Albert had no fear of the rat, he was interested in the rat and wanted to play with it.
Unconditioned Stimulus (US): This is a stimulus that naturally and automatically triggers a response without any learning. In the experiment, the unconditioned stimulus was the loud, frightening noise. This noise was produced by Watson and Rayner striking a steel bar with a hammer behind Albert’s back.
Unconditioned Response (UR): This is the natural response that occurs when the Unconditioned Stimulus is presented. It is unlearned and occurs without previous conditioning. In this case, the Unconditioned Response was Albert’s fear response to the loud noise – crying and showing distress.
Conditioning Process: Watson and Rayner then began the conditioning process. They presented the rat (NS) to Albert, and then, while he was interacting with the rat, they made a loud noise (US). This was done repeatedly, pairing the sight of the rat with the frightening noise. As a result, Albert started associating the rat with the fear he experienced due to the loud noise.
Conditioned Stimulus (CS): After several pairings, the previously Neutral Stimulus (the rat) becomes the conditioned stimulus , as it now elicits the fear response even without the presence of the loud noise.
Conditioned Response (CR): This is the learned response to the previously neutral stimulus, which is now the Conditioned Stimulus. In this case, the Conditioned Response was Albert’s fear of the rat. Even without the loud noise, he became upset and showed signs of fear whenever he saw the rat.

In this experiment, a previously unafraid baby was conditioned to become afraid of a rat. It also demonstrates two additional concepts, originally outlined by Pavlov .

Extinction : Although a conditioned association can be incredibly strong initially, it begins to fade if not reinforced – until is disappears completely.
Generalization : Conditioned associations can often widen beyond the specific stimuli presented. For instance, if a child develops a negative association with one teacher, this association might also be made with others.

Over the next few weeks and months, Little Albert was observed and ten days after conditioning his fear of the rat was much less marked. This dying out of a learned response is called extinction.

However, even after a full month, it was still evident, and the association could be renewed by repeating the original procedure a few times.

Unfortunately, Albert’s mother withdrew him from the experiment the day the last tests were made, and Watson and Rayner were unable to conduct further experiments to reverse the condition response.

The Little Albert experiment was a controversial psychology experiment by John B. Watson and his graduate student, Rosalie Rayner, at Johns Hopkins University.
The experiment was performed in 1920 and was a case study aimed at testing the principles of classical conditioning.
Watson and Raynor presented Little Albert (a nine-month-old boy) with a white rat, and he showed no fear. Watson then presented the rat with a loud bang that startled Little Albert and made him cry.
After the continuous association of the white rat and loud noise, Little Albert was classically conditioned to experience fear at the sight of the rat.
Albert’s fear generalized to other stimuli that were similar to the rat, including a fur coat, some cotton wool, and a Santa mask.

Critical Evaluation

Methodological limitations.

The study is often cited as evidence that phobias can develop through classical conditioning. However, critics have questioned whether conditioning actually occurred due to methodological flaws (Powell & Schmaltz, 2022).

The study didn’t control for pseudoconditioning – the loud noise may have simply sensitized Albert to be fearful of any novel stimulus.
It didn’t control for maturation – Albert was 11 months old initially, but the final test was at 12 months. Fears emerge naturally over time in infants, so maturation could account for Albert’s reactions.
Albert’s reactions were inconsistent and the conditioned fear weak – he showed little distress to the rat in later tests, suggesting the conditioning was not very effective or durable.

Other methodological criticisms include:

The researchers confounded their own experiment by conditioning Little Albert using the same neutral stimuli as the generalized stimuli (rabbit and dog).
Some doubts exist as to whether or not this fear response was actually a phobia. When Albert was allowed to suck his thumb he showed no response whatsoever. This stimulus made him forget about the loud sound. It took more than 30 times for Watson to finally take Albert’s thumb out to observe a fear response.
Other limitations included no control subject and no objective measurement of the fear response in Little Albert (e.g., the dependent variable was not operationalized).
As this was an experiment of one individual, the findings cannot be generalized to others (e.g., low external validity). Albert had been reared in a hospital environment from birth and he was unusual as he had never been seen to show fear or rage by staff. Therefore, Little Albert may have responded differently in this experiment to how other young children may have, these findings will therefore be unique to him.

Theoretical Limitations

The cognitive approach criticizes the behavioral model as it does not take mental processes into account. They argue that the thinking processes that occur between a stimulus and a response are responsible for the feeling component of the response.

Ignoring the role of cognition is problematic, as irrational thinking appears to be a key feature of phobias.

Tomarken et al. (1989) presented a series of slides of snakes and neutral images (e.g., trees) to phobic and non-phobic participants. The phobics tended to overestimate the number of snake images presented.

The Little Albert Film

Powell and Schmaltz (2022) examined film footage of the study for evidence of conditioning. Clips showed Albert’s reactions during baseline and final transfer tests but not the conditioning trials. Analysis of his reactions did not provide strong evidence of conditioning:

With the rat, Albert was initially indifferent and tried to crawl over it. He only cried when the rat was placed on his hand, likely just startled.
With the rabbit, dog, fur coat, and mask, his reactions could be explained by being startled, innate wariness of looming objects, and other factors. Reactions were inconsistent and mild.

Overall, Albert’s reactions seem well within the normal range for an infant and can be readily explained without conditioning. The footage provides little evidence he acquired conditioned fear.

The belief the film shows conditioning may stem from:

Viewer expectation – titles state conditioning occurred and viewers expect to see it.
A tendency to perceive stronger evidence of conditioning than actually exists.
An ongoing perception of behaviorism as manipulative, making Watson’s conditioning of a “helpless” infant seem plausible.

Rather than an accurate depiction, the film may have been a promotional device for Watson’s research. He hoped to use it to attract funding for a facility to closely study child development.

This could explain anomalies like the lack of conditioning trials and rearrangement of test clips.

Ethical Issues

The Little Albert Experiment was conducted in 1920 before ethical guidelines were established for human experiments in psychology. When judged by today’s standards, the study has several concerning ethical issues:

There was no informed consent obtained from Albert’s parents. They were misled about the true aims of the research and did not know their child would be intentionally frightened. This represents a lack of transparency and a violation of personal autonomy.
Intentionally inducing a fear response in an infant is concerning from a nonmaleficence perspective, as it involved deliberate psychological harm. The distress exhibited by Albert suggests the conditioning procedure was unethical by today’s standards.
Watson and Rayner did not attempt to decondition or desensitize Albert to the fear response before the study ended abruptly. This meant they did not remove the psychological trauma they had induced, violating the principle of beneficence. Albert was left in a state of fear, which could have long-lasting developmental effects. Watson also published no follow-up data on Albert’s later emotional development.

Learning Check

Summarise the process of classical conditioning in Watson and Raynor’s study.
Explain how Watson and Raynor’s methodology is an improvement on Pavlov’s.
What happened during the transfer sessions? What did this demonstrate?
Why is Albert’s reaction to similar furry objects important for the interpretation of the study?
Comment on the ethics of Watson and Raynor’s study.
Support the claim that in ignoring the internal processes of the human mind, behaviorism reduces people to mindless automata (robots).

Beck, H. P., Levinson, S., & Irons, G. (2009). Finding Little Albert: A journey to John B. Watson’s infant laboratory. American Psychologist, 64 , 605–614.

Digdon, N., Powell, R. A., & Harris, B. (2014). Little Albert’s alleged neurological persist impairment: Watson, Rayner, and historical revision. History of Psychology , 17 , 312–324.

Fridlund, A. J., Beck, H. P., Goldie, W. D., & Irons, G. (2012). Little Albert: A neurologically impaired child. History of Psychology , 15, 1–34.

Griggs, R. A. (2015). Psychology’s lost boy: Will the real Little Albert please stand up? Teaching of Psychology, 4 2, 14–18.

Harris, B. (1979). Whatever happened to little Alb ert? . American Psychologist, 34 (2), 151.

Harris, B. (2011). Letting go of Little Albert: Disciplinary memory, history, and the uses of myth. Journal of the History of the Behavioral Sciences, 47 , 1–17.

Harris, B. (2020). Journals, referees and gatekeepers in the dispute over Little Albert, 2009–2014. History of Psychology, 23 , 103–121.

Powell, R. A., Digdon, N., Harris, B., & Smithson, C. (2014). Correcting the record on Watson, Rayner, and Little Albert: Albert Barger as “psychology’s lost boy.” American Psychologist, 69 , 600–611.

Powell, R. A., & Schmaltz, R. M. (2021). Did Little Albert actually acquire a conditioned fear of furry animals? What the film evidence tells us. History of Psychology , 24 (2), 164.

Todd, J. T. (1994). What psychology has to say about John B. Watson: Classical behaviorism in psychology textbooks. In J. T. Todd & E. K. Morris (Eds.), Modern perspectives on John B. Watson and classical behaviorism (pp. 74–107). Westport, CT: Greenwood Press.

Tomarken, A. J., Mineka, S., & Cook, M. (1989). Fear-relevant selective associations and covariation bias. Journal of Abnormal Psychology, 98 (4), 381.

Watson, J.B. (1913). Psychology as the behaviorist Views It. Psychological Review, 20 , 158-177.

Watson, J. B., & Rayner, R. (1920). Conditioned emotional reactions . Journal of Experimental Psychology, 3 (1), 1.

Watson, J. B., & Watson, R. R. (1928). Psychological care of infant and child . New York, NY: Norton.

Further Information

Finding Little Albert
Mystery solved: We now know what happened to Little Albert
Psychology’s lost boy: Will the real Little Albert please stand up?
Journals, referees, and gatekeepers in the dispute over Little Albert, 2009-2014
Griggs, R. A. (2014). The continuing saga of Little Albert in introductory psychology textbooks. Teaching of Psychology, 41(4), 309-317.

Rodent Models of Conditioned Fear: Behavioral Measures of Fear and Memory

First Online: 01 January 2012

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Jennifer L. McGuire 4 ,
Jennifer L. Coyner 4 , 5 &
Luke R. Johnson 4 , 6 , 5

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

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Pavlovian fear conditioning is a robust technique for examining behavioral and cellular components of fear learning and memory. In fear conditioning, the subject learns to associate a previously neutral stimulus with an inherently noxious co-stimulus. The learned association is reflected in the subjects’ behavior upon subsequent re-exposure to the previously neutral stimulus or the training environment. Using fear conditioning, investigators can obtain a large amount of data that describe multiple aspects of learning and memory. In a single test, researchers can evaluate functional integrity in fear circuitry, which is both well characterized and highly conserved across species. Additionally, the availability of sensitive and reliable automated scoring software makes fear conditioning amenable to high-throughput experimentation in the rodent model; thus, this model of learning and memory is particularly useful for pharmacological and toxicological screening. Due to the conserved nature of fear circuitry across species, data from Pavlovian fear conditioning are highly translatable to human models. We describe equipment and techniques needed to perform and analyze conditioned fear data. We provide two examples of fear conditioning experiments, one in rats and one in mice, and the types of data that can be collected in a single experiment.

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Fear Conditioning in Laboratory Rodents

Sound check, stage design and screen plot – how to increase the comparability of fear conditioning and fear extinction experiments

Fear Models in Animals and Humans

LeDoux JE (2000) Emotion circuits in the brain. Ann Rev Neurosci 23:155–184

Article CAS PubMed Google Scholar

Johnson LR et al (2008) A recurrent network in the lateral amygdala: a mechanism for coincidence detection. Front Neural Circuits 2:3

Article PubMed Central PubMed Google Scholar

Maren SQ, Quirk GJ (2004) Neuronal signaling of fear and memory. Neuroscience 5:844–852

CAS PubMed Google Scholar

Blair H et al (2001) Synaptic plasticity in the lateral amygdala: a cellular hypothesis. Learn Mem 8:229–242

Johansen JP et al (2011) Molecular mechanisms of fear learning and memory. Cell 147(3):509–524

Article CAS PubMed Central PubMed Google Scholar

Choi JS, Brown TH (2003) Central amygdala lesions block ultrasonic vocalization and freezing as conditional but not unconditional responses. J Neurosci 23(25):8713–8721

Ciocchi S et al (2010) Encoding of conditioned fear in central amygdala inhibitory circuits. Nature 468(7321):277–282

Wilensky AE et al (2006) Rethinking the fear circuit: the central nucleus of the amygdala is required for the acquisition, consolidation, and expression of Pavlovian fear conditioning. J Neurosci 26(48):12387–12396

Pare D, Quirk GJ, Ledoux JE (2004) New vistas on amygdala networks in conditioned fear. J Neurophysiol 92(1):1–9

Article PubMed Google Scholar

Fanselow MS, LeDoux JE (1999) Why we think plasticity underlying Pavlovian fear conditioning occurs in the basolateral amygdala. Neuron 23(2):229–232

Maren S, Aharonov G, Fanselow MS (1997) Neurotoxic lesions of the dorsal hippocampus and Pavlovian fear conditioning in rats. Behav Brain Res 88(2):261–274

Phillips RG, LeDoux JE (1992) Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behav Neurosci 106(2):274–285

Vidal-Gonzalez I et al (2006) Microstimulation reveals opposing influences of prelimbic and infralimbic cortex on the expression of conditioned fear. Learn Mem 13(6):728–733

Milad MR, Quirk GJ (2002) Neurons in medial prefrontal cortex signal memory for fear extinction. Nature 420(6911):70–74

Morgan MA, LeDoux JE (1995) Differential contribution of dorsal and ventral medial prefrontal cortex to the acquisition and extinction of conditioned fear in rats. Behav Neurosci 109(4):681–688

Sotres-Bayon F, Quirk GJ (2010) Prefrontal control of fear: more than just extinction. Curr Opin Neurobiol 20(2):231–235

Corcoran KA, Quirk GJ (2007) Activity in prelimbic cortex is necessary for the expression of learned, but not innate, fears. J Neurosci 27(4):840–844

Stiedl O et al (2004) Behavioral and autonomic dynamics during contextual fear conditioning in mice. Auton Neurosci 115(1–2):15–27

Brinks V, de Kloet ER, Oitzl MS (2008) Strain specific fear behaviour and glucocorticoid response to aversive events: modeling PTSD in mice. Prog Brain Res 167:257–261

Rodrigues SL (2009) JE; Sapolsky, RM, The influence of stress hormones on fear circuitry. Ann Rev Neurosci 32:289–313

Bronstein PM, Hirsch SM (1976) Ontogeny of defensive reactions in Norway rats. J Comp Physiol Psychol 90(7):620–629

Clinchy M et al (2010) The neurological ecology of fear: insights neuroscientists and ecologists have to offer one another. Front Behav Neurosci 4:21

PubMed Google Scholar

Blanchard RJ, Blanchard DC (1969) Passive and active reactions to fear-eliciting stimuli. J Comp Physiol Psychol 68(1):6

Article Google Scholar

Blanchard RJ, Flannelly KJ, Blanchard DC (1986) Defensive behavior of laboratory and wild Rattus norvegicus. J Comp Physiol Psychol 100(2):6

Stanton ME (2000) Multiple memory systems, development and conditioning. Behav Brain Res 110(1–2):25–37

Prager EM et al (2011) The importance of reporting housing and husbandry in rat research. Front Behav Neurosci 5:38

Sharp JL et al (2002) Stress-like responses to common procedures in male rats housed alone or with other rats. Contemp Top Lab Anim Sci 41(4):8–14

Google Scholar

Voikar V et al (2005) Long-term individual housing in C57BL/6J and DBA/2 mice: assessment of behavioral consequences. Genes Brain Behav 4(4):240–252

Mitra RA, Adamec R, Sapolsky R (2009) Resilience against predator stress and dendritic morphology of amygdala neurons. Behav Brain Res 205:535–543

Barbelivien A et al (2006) Environmental enrichment increases responding to contextual cues but decreases overall conditioned fear in the rat. Behav Brain Res 169(2):231–238

Kopec CD et al (2007) A robust automated method to analyze rodent motion during fear conditioning. Neuropharmacology 52(1):228–233

Schmitt U, Hiemke C (1998) Strain differences in open-field and elevated plus-maze behavior of rats without and with pretest handling. Pharmacol Biochem Behav 59(4):807–811

Longordo F et al (2011) Do mice habituate to “gentle handling?” A comparison of resting behavior, corticosterone levels and synaptic function in handled and undisturbed C57BL/6J mice. Sleep 34(5):679–681

Bolles C, Collier A (1976) Effect of predictive cues on freezing in rats. Anim Learn Behav 4:2

Chaudhury D, Colwell CS (2002) Circadian modulation of learning and memory in fear-conditioned mice. Behav Brain Res 133(1):95–108

Eckel-Mahan KL et al (2008) Circadian oscillation of hippocampal MAPK activity and cAmp: implications for memory persistence. Nat Neurosci 11(9):1074–1082

Gerstner JR et al (2009) Cycling behavior and memory formation. J Neurosci 29(41):12824–12830

Kandel ER (2001) The molecular biology of memory storage: a dialogue between genes and synapses. Science 294(5544):1030–1038

Schafe GE, LeDoux JE (2000) Memory consolidation of auditory Pavlovian fear conditioning requires Protein synthesis and protein kinase A in the amygdala. J Neurosci 20(18):Rc96

Schafe GE et al (2001) Memory consolidation of Pavlovian fear conditioning: a cellular and molecular perspective. Trends Neurosci 24(9):540–546

Izquierdo I et al (1999) Separate mechanisms for short- and long-term memory. Behav Brain Res 103(1):1–11

Schafe GE et al (1999) Memory consolidation for contextual and auditory fear conditioning is dependent on protein synthesis, PKA, and MAP kinase. Learn Mem 6(2):97–110

Barondes SH, Cohen HD (1967) Delayed and sustained effect of acetoxycycloheximide on memory in mice. Proc Natl Acad Sci USA 58(1):157–164

Lubow RE, Moore AU (1959) Latent inhibition: the effect of nonreinforced pre-exposure to the conditional stimulus. J Comp Physiol Psychol 52:415–419

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McGuire, J.L., Coyner, J.L., Johnson, L.R. (2012). Rodent Models of Conditioned Fear: Behavioral Measures of Fear and Memory. In: Szallasi, A., Bíró, T. (eds) TRP Channels in Drug Discovery. Methods in Pharmacology and Toxicology. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-095-3_11

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December 1, 2013

Fearful Memories Passed Down to Mouse Descendants

Genetic imprint from traumatic experiences carries through at least two generations

By Ewen Callaway & Nature magazine

From Nature magazine

Certain fears can be inherited through the generations, a provocative study of mice reports. The authors suggest that a similar phenomenon could influence anxiety and addiction in humans. But some researchers are sceptical of the findings because a biological mechanism that explains the phenomenon has not been identified.

According to convention, the genetic sequences contained in DNA are the only way to transmit biological information across generations. Random DNA mutations, when beneficial, enable organisms to adapt to changing conditions, but this process typically occurs slowly over many generations.

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Yet some studies have hinted that environmental factors can influence biology more rapidly through 'epigenetic' modifications, which alter the expression of genes, but not their actual nucleotide sequence. For instance, children who were conceived during a harsh wartime famine in the Netherlands in the 1940s are at increased risk of diabetes, heart disease and other conditions — possibly because of epigenetic alterations to genes involved in these diseases. Yet although epigenetic modifications are known to be important for processes such as development and the inactivation of one copy of the X-chromsome in females, their role in the inheritance of behaviour is still controversial.

Kerry Ressler, a neurobiologist and psychiatrist at Emory University in Atlanta, Georgia, and a co-author of the latest study, became interested in epigenetic inheritance after working with poor people living in inner cities, where cycles of drug addiction, neuropsychiatric illness and other problems often seem to recur in parents and their children. “There are a lot of anecdotes to suggest that there’s intergenerational transfer of risk, and that it’s hard to break that cycle,” he says.

Heritable traits

Studying the biological basis for those effects in humans would be difficult. So Ressler and his colleague Brian Dias opted to study epigenetic inheritance in laboratory mice trained to fear the smell of acetophenone, a chemical the scent of which has been compared to those of cherries and almonds. He and Dias wafted the scent around a small chamber, while giving small electric shocks to male mice. The animals eventually learned to associate the scent with pain, shuddering in the presence of acetophenone even without a shock.

This reaction was passed on to their pups, Dias and Ressler report today in Nature Neuroscience 1. Despite never having encountered acetophenone in their lives, the offspring exhibited increased sensitivity when introduced to its smell, shuddering more markedly in its presence compared with the descendants of mice that had been conditioned to be startled by a different smell or that had gone through no such conditioning. A third generation of mice — the 'grandchildren' — also inherited this reaction, as did mice conceived through in vitro fertilization with sperm from males sensitized to acetophenone. Similar experiments showed that the response can also be transmitted down from the mother.

These responses were paired with changes to the brain structures that process odours. The mice sensitized to acetophenone, as well as their descendants, had more neurons that produce a receptor protein known to detect the odour compared with control mice and their progeny. Structures that receive signals from the acetophenone-detecting neurons and send smell signals to other parts of the brain (such as those involved in processing fear) were also bigger.

The researchers propose that DNA methylation — a reversible chemical modification to DNA that typically blocks transcription of a gene without altering its sequence — explains the inherited effect. In the fearful mice, the acetophenone-sensing gene of sperm cells had fewer methylation marks, which could have led to greater expression of the odorant-receptor gene during development.

But how the association of smell with pain influences sperm remains a mystery. Ressler notes that sperm cells themselves express odorant receptor proteins, and that some odorants find their way into the bloodstream, offering a potential mechanism, as do small, blood-borne fragments of RNA known as microRNAs, that control gene expression.

Contentious findings

Predictably, the study has divided researchers. “The overwhelming response has been 'Wow! But how the hell is it happening?'" says Dias. David Sweatt, a neurobiologist at the University of Alabama at Birmingham who was not involved in the work, calls it “the most rigorous and convincing set of studies published to date demonstrating acquired transgenerational epigenetic effects in a laboratory model".

However, Timothy Bestor, a molecular biologist at Columbia University in New York who studies epigenetic modifications, is incredulous. DNA methylation is unlikely to influence the production of the protein that detects acetophenone, he says. Most genes known to be controlled by methylation have these modifications in a region called the promoter, which precedes the gene in the DNA sequence. But the acetophenone-detecting gene does not contain nucleotides in this region that can be methylated, Bestor says. "The claims they make are so extreme they kind of violate the principle that extraordinary claims require extraordinary proof,” he adds.

Tracy Bale, a neuroscientist at the University of Pennsylvania in Philadelphia, says that researchers need to “determine the piece that links Dad's experience with specific signals capable of producing changes in epigenetic marks in the germ cell, and how these are maintained”.

“It's pretty unnerving to think that our germ cells could be so plastic and dynamic in response to changes in the environment,” she says.

Humans inherit epigenetic alterations that influence behaviour, too, Ressler suspects. A parent’s anxiety, he speculates, could influence later generations through epigenetic modifications to receptors for stress hormones. But Ressler and Dias are not sure how to prove the case, and they plan to focus on lab animals for the time being.

The researchers now want to determine for how many generations the sensitivity to acetophenone lasts, and whether that response can be eliminated. Scepticism that the inheritance mechanism is real will likely persist, Ressler says, “until someone can really explain it in a molecular way”, says Ressler. “Unfortunately, it’s probably going to be complicated and it’s probably going to take a while.”

This article is reprinted with permission from Nature magazine. It was first published on December 1, 2013.

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Conditioned turning behavior: A Pavlovian fear response expressed during the post-encounter period following aversive stimulation

Rats were trained to fear an auditory conditioned stimulus (CS) by pairing it with a mild electric shock (the unconditioned stimulus, or US) delivered to one eyelid. After training, the CS elicited two different conditioned fear responses from rats: a passive freezing response, and an active turning response. The balance between these two modes of conditioned responding depended upon the rat's recent history of encounters with the US. If rats had not recently encountered the US, then they responded to the CS by freezing. But after recently encountering the US, rats exhibited CS-evoked turning responses that were always directed away from the trained eyelid, even if the US had recently been delivered to the opposite (untrained) eyelid. This post-encounter turning behavior was not observed in rats that had been trained with unpaired presentations of the CS and US, indicating that even though CS-evoked turning was selectively expressed after recent encounters with the US, it was nonetheless a conditioned Pavlovian fear response that depended upon a learned association between the CS and US. Further supporting this conclusion, pharmacological inactivation experiments showed that expression of both freezing and turning behaviors depended upon lateralized circuits in the amygdala and periaqueductal gray (PAG) that are known to support expression of Pavlovian fear responses. These findings indicate that even though the ability of a CS to elicit Pavlovian fear responses depend upon the long-term history of CS-US pairings, the mode of conditioned responding (freezing versus turning in the present experiments) can be modulated by short-term factors, such as the recent history of US encounters. We discuss neural mechanisms that might mediate such short-term transitions between different modes of defensive responding, and consider how dysregulation of such mechanisms might contribute to clinical anxiety disorders.

INTRODUCTION

Most animals (including humans) are endowed with an innate repertoire of defensive responses for coping with threats to their survival. Defensive responses change as threat levels increase, and can thus be organized along a spectrum referred to as the “predatory imminence continuum” ( Blanchard & Blanchard, 1969a , b ; Bolles, 1970 ; Fanselow & Lester, 1988 ; Mobbs et al., 2007 , 2009 ). In rats, low levels of threat are characterized by engagement in non-defensive behaviors, such as exploration or goal-seeking. At intermediate threat levels (referred to as “circa-strike”), the rat begins to perceive danger and engage in behaviors such as freezing to avoid detection by potential predators, or emitting warning calls to notify conspecifics of a possible threat. The highest threat levels (referred to as “post-encounter”) occur after the rat has suffered injury or come under attack by a predator, triggering responses such as distress calls, fleeing from danger, or fighting back against the predator if no escape is possible.

Much of what is currently known about neural circuits mediating defensive responses has been learned from rodent studies of Pavlovian fear conditioning, in which rats (or mice) are trained to fear a neutral CS by pairing it with an aversive US (for review see Davis, 1992 ; LeDoux, 2000 ). In such studies, conditioned fear is typically assessed by presenting a CS to previously trained subjects that have not recently encountered the US, while they are in a baseline state of low predatory imminence (for example, freely exploring their environment, or engaged in a task such as licking or bar-pressing). Under these testing conditions, the CS can elicit circa-strike defenses—such as freezing or startle potentiation—which are measured to index the level of conditioned fear. An underlying assumption of such experiments is that expression of the measured responses is monotonically related to the intensity of conditioned fear (that is, more responding indicates more fear). However, this monotonicity assumption may not always be valid, because if fear intensity exceeds the threshold for triggering post-encounter defensive strategies, then decreases in the expression of circa-strike behaviors (like freezing or startle) may reflect greater—not lesser—fear of the CS (see Blanchard & Blanchard, 1969a , b ; Bolles, 1970 ; Davis & Astrachan, 1979; Fanselow & Lester, 1988 ). Consequently, the range of conditioned fear intensities that can be accurately indexed by Pavlovian circa-strike behaviors is constrained to remain below the threshold for expression of post-encounter defenses. This is an unfortunate limitation, because rodent fear conditioning has been widely adopted as an animal model for investigating the neurobiological basis of clinical anxiety disorders ( Davis and Whalen, 2001 ; Rau et al., 2005 ; Davis et al., 2006 ; Milad et al. 2006 ; Miller & McEwen, 2006 ; Rauch et al., 2006 ). But some anxiety symptoms in human patients—such as panic attacks—may involve activation of post-encounter response systems (see Craske, 1999 ). Standard rodent models of fear conditioning may not recruit these post-encounter response systems, since they are based upon methods that favor the expression of circa-strike behaviors.

We have previously conducted fear conditioning experiments using a paradigm in which rats are given an auditory CS paired with a unilateral shock US delivered to one eyelid ( Moita et al., 2003 , 2004 ; Blair et al., 2005a , b ; Tarpley et al. 2009 ; Johansen et al. 2010 ). During these experiments, we have observed that in addition to CS-evoked freezing behavior, well-trained rats also tend to exhibit another distinctive response to the CS: turning in circles away from the eyelid where shock is anticipated. Here, we conducted a formal investigation of this novel turning response. We report that, like freezing, CS-evoked turning behavior is a Pavlovian response which depends upon lateralized circuits in the amygdala and PAG that mediate acquisition and expression of conditioned fear ( Fanselow, 1991 ; Bandler & DePaulis, 1991 ; Davis, 1992 ; Maren, 1998; LeDoux, 2000 ). But unlike freezing, the turning response is expressed selectively after recent encounters with the US, and not at other times. These results suggest that conditioned turning responses may be expressed selectively when the intensity of conditioned fear exceeds the threshold for triggering post-encounter defenses, which does not occur unless the US has recently been encountered. Based on these findings, we propose that conditioned turning responses may provide a useful behavioral index for investigating clinically relevant questions concerning neural substrates that mediate post-encounter defensive strategies.

All experimental procedures were approved by the UCLA Animal Research Committee and were conducted in accordance with U.S. federal guidelines for animal research.

Subjects and surgery

Male Long-Evans rats weighing 350–400 g were housed singly and reduced to 85% of ad-lib weight through limited daily feeding. Under deep isoflourane anesthesia, all but two rats (see below) were implanted with a pair of very thin insulated stainless steel wires (75 μm diameter) threaded into the skin of each eyelid for delivering the mild periorbital shock US. Rats in the experimental groups were implanted with a pair of 26 gauge microinjector guide cannulae (Plastics One, Roanoake, VA) targeted bilaterally in the lateral nucleus of the amygdala (3.0 mm posterior, 5.3 mm lateral and 8.0 mm ventral to bregma) or PAG (7.8 mm posterior, 0.75 mm lateral and 5.8 mm ventral to bregma). All implants were secured in place with bone cement and anchoring screws. At the conclusion of the surgery, rats were removed from the stereotaxic frame and observed until they fully emerged from anesthesia, then retured to their home cages and allowed to recover for at least 5 days prior to beginning experiments. Two rats (one implanted in the amgdala, the other in PAG) were not implanted with periorbital stimulus wires, and were not removed from the stereotaxic frame at the end of the surgery, but instead were given intracranial infusions (0.4 μl a rate of 0.25 μl /min) of fluorescent muscimol (tagged with Bodipy® TMR-X fluorophore, Invitrogen product #M2400), dissolved at 0.25 mg/ml in sterile 0.9% saline vehicle (this was the same volume, concentration, and rate used for infusions of non-flourescent muscimol in behavioural experiments, see below). 30 min after the infusion was completed, rats were removed from the stereotaxic frame, euthanized with an intraperitoneal overdose of pentobarbital (100 mg/kg), and transcardially perfused with formalin so that brain tissue could be prepared for histological analysis of muscimol diffusion.

Fear conditioning experiments

After recovery from surgery, rats were pre-exposed for 5 days (15 min/day) to the experimental platform before any fear conditioning sessions were conducted. Throughout pre-exposure and fear conditioning sessions, rats constantly foraged on a 70×70 cm platform for 20 mg purified food pellets (Bioserv, Frenchtown, NJ) dropped from an overhead dispenser at ~30 s intervals, to provide a baseline of motor activity against which stimulus-evoked freezing, movement, and turning behavior could be measured. The CS for fear conditioning was a train of 70 dB white noise pips, each lasting 250 ms, delivered at 1 Hz for 20 s through an overhead speaker. The US was a train of very brief 2.0 mA shock pulses, each lasting 2.0 ms, delivered to the skin above one eyelid at a rate of 6.66 Hz for 2 s. During CS-US pairing trials, the first shock pulse was always delivered 300 ms after the offset of the final (20 th ) CS pip. The interval between CS onset of successive trials was uniformly random between 180 and 240 s for all testing and training trials. Rats implanted with AMG cannulae were trained drug-free for 7 days prior to their first intracranial infusion, whereas rats implanted with PAG cannulae were trained drug-free for 4 days prior to their first infusion.

Rats in the unpaired control group were trained with explicitly unpaired presentations of the CS and US, by delivering the US exactly halfway between CS onset of successive trials (which were separated by a uniformly random interval of 180 and 240 s, as in paired training). In studies of Pavlovian conditioning, it is usually preferable to randomize the order of CS and US alone trials in the unpaired controls. But here, the conditioned response under study (CS-evoked turning) was strongly modulated by the recency of US delivery. This made it necessary to preserve the alternating order of CS and US presentations in both the paired and unpaired groups, because presenting several CS alone trials in a row to unpaired rats (which would sometimes occur with a randomized trial order) could diminish the CS-evoked turning response by increasing the separation between the CS and the most recent US, and thus reduce conditioned responding by mechanisms unrelated to associative learning. Explicitly unpaired presentations of the CS and US—as we have used here—can cause the CS to acquire properties of a conditioned inhibitor ( Rescorla, 1969 ), and this potential confound is addressed in the Results section.

Behavioral Scoring

The rat's moment-to-moment position on the platform was sampled at 30 Hz by an overhead video tracking system (Neuralynx Corporation, Bozeman, MT), which monitored the location of three light-emitting diodes (red, blue, green) attached to the animal's headstage for automated scoring of freezing, movement, and turning behavior using software developed in our laboratory. The algorithm for scoring freezing behavior has been described elsewhere ( Moita et al., 2003 ; 2004 ). The algorithm for scoring movement and turning behavior first performed one iteration of smoothing (5-point adjacent averaging) upon the position data for each of the three colored LED's. The center point of the three LEDs was obtained by averaging their x and y coordinates, and the displacement distance of this center position between each successive video frame gave the rat's linear movement speed. The angles of the axes passing through each pair of tracking LED's (red-green, red-blue, blue-green) was measured with respect to the horizontal axis of the video screen. Two of the angles (red-blue and blue-green) were rotated by the appropriate amount to align them with the third angle (red-green), and the mean of these transformed angular measurements was computed using circular averaging to obtain the rat's directional heading for each video frame (if one of the LEDs was occluded, then only one of the three color axes was used to estimate the directional heading). The change in directional heading angle between each successive video frame gave the rat's angular head-turning velocity.

Muscimol Infusions

Muscimol was dissolved at a concentration of 0.25 mg/ml in sterile 0.9% saline vehicle, and infused intracranially at a rate of 0.25 μl /min. For both amygdala and PAG infusions, a volume of 0.4 μl was infused into each hemisphere through 33-gauge injectors. Prior to drug infusion, dummy cannulae (which were in place at all times except during infusions, to prevent clogging of the guide cannulae) were removed and injector cannulae were inserted in their place. After drug infusion, the injectors were left in place for an additional 1–2 min to allow diffusion of the drug away from the cannulae tip, after which the injectors were removed and replaced with dummy cannulae. Throughout the infusion process, the animal was held gently on the experimenter's lap. After the infusion was complete, the rat was returned to its home cage for 15 min to allow time for the drug to take effect before the experiment resumed. At the conclusion of the experiment, rats were euthanized by intrapertoneally injected overdose of pentobarbital (100 mg/kg), and trancardially perfused with formalin so that the brain tissue could be removed and sectioned to verify the placements of the injector tips (results shown in Figures 3A and 3B ).

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Reconstructed placements of infusion cannula for the AMG (panel A) and PAG (panel B) groups are shown using templates from the atlas of Paxinos and Watson (1997) . Hemispheres are labeled ipsilateral (IPSI) versus contralateral (CONTRA) with respect to the trained eyelid for each rat. Panel C shows imaging of fluorescent muscimol (red) against DAPI counterstain (blue) to illustrate diffusion of the drug away from injection sites in amygdala and PAG.

Experiment 1: Characterization of conditioned turning responses

Experimental design.

Three groups of rats underwent fear conditioning: 1) an amygdala (AMG) group (n=14) that was bilaterally implanted with intracranial infusion cannulae in the lateral nucleus of the amygdala, 2) a PAG group (n=10) that was bilaterally implanted with infusion cannulae in the lateral and ventrolateral columns of PAG, and 3) an unpaired (UNP) control group (n=10) which was implanted only with periorbital stimulus wires for US delivery, but not with any infusion cannula. On each day of the experiment following initial pre-exposure sessions (see Methods), rats first received 6 test trials in which the CS was presented alone without the US. Following these test trials (after a standard intertrial interval of 180–240 s), rats in the experimental groups (AMG and PAG) received 16 CS-US pairings, while rats in the UNP group received 16 explicitly unpaired presentations of the CS and US. In the PAG and UNP groups, half of the rats received the US on the left eyelid, and the other half on the right. In the AMG group, the US was delivered to the left eyelid of 8 rats and the right eyelid of 6 rats.

Acquisition of conditioned freezing responses

A standard regimen of 6 test trials followed by 16 training trials was given to all three groups of rats on four consecutive days of acquisition training. Since the AMG and PAG groups were trained identically with CS-US pairings on these four acquisition days, the rats in these two groups were combined into a single group designated as the PAIR group (n=24), which was compared against the UNP group to evaluate the effects of paired versus unpaired CS-US presentations upon the acquisition of condition defensive responses.

Figure 1A plots averaged freezing behavior (measured by the amount of time when the headstage tracking lights were immobile) during the context (CX) and CS periods on each trial across days 1–4 of the experiment. The immobility scores from each day were analyzed using a 2×2×2 ANOVA with stimulus (CX vs. CS) and trial type (test vs. training) as repeated factors, and group (PAIR vs. UNP) as an independent factor. The three-way interaction effect (stimulus × trial type × group) was significant on every day after the first day of training (day 1: F 1,32 =2.03, p=.17; day 2: F 1,32 =35.3, p<.00001; day 3: F 1,32 =27.2, p=.00001; day 4: F 1,32 =25.0, p=.00002), so all of the factors appeared to influence freezing behavior in trained rats. Newman-Keuls posthoc comparisons revealed that on every day after the first day, rats in the PAIR group froze more during the CS than the CX during test trials (day 1: p=.21; day 2: p=.0003; day 3: p=.06; day 4: p=0006; note that CS-evoked freezing did not quite reach statistical significance on day 3). By contrast, rats in the UNP group never froze more to the CS than the CX during test trials (day 1: p=.58; day 2: p=.44; day 3: p=.75; day 4: p=.63). These results agree well with prior studies showing that when an auditory CS has been paired with an eyelid shock US, the CS elicits conditioned freezing responses when it is later presented alone without the US ( Moita et al., 2003 ; Lee and Kim, 2004 ; Blair et al., 2005a , b ). Thus, based on the expression of freezing behavior during test trials ( Figure 1A , shaded regions), it appears that rats in the PAIR but not the UNP group acquired conditioned freezing responses to the CS, as expected.

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Each graph shows averaged behavioral responses over 6 test trials (gray shaded regions) and 16 training trials (unshaded regions) on a single day of the experiment. A) Freezing behavior, measured as the amount of time the headstage tracking lights were immobile during the trial. B) Head movement speed, measured as the mean speed of the headstage lights (in cm/s) during the trial. C) Head turning velocity, measured as the speed of angular rotation (in deg/s) of the headstage lights; positive values indicate turning in the direction away from the shocked eyelid, whereas negative values indicate turning toward the shocked eyelid.

However, the expression of CS-evoked freezing responses during test trials was abruptly reversed during the training trials that followed the test trials on each day. Instead of freezing more to the CS than the CX, rats in the PAIR group froze more to the CX than the CS during training trials ( Figure 1A , unshaded regions). This reversal of behavior was evident in the fact that in the PAIR group, the 2×2 interaction between stimulus (CX vs. CS) and session (test vs. training) was significant on all four days (day 1: F 1,23 =7.52, p=.01; day 2: F 1,23 =81.6, p<.00001; day 3: F 1,23 =38.3, p<.00001; day 4: F 1,23 =43.4, p<.00001), and freezing to the CX was significantly greater than to the CS during training sessions on days 2–4 (day 1: F 1,9 =3.41, p=.08; day 2: F 1,9 =27.6, p=.00003; day 3: F 1,9 =16.2, p=.04; day 4: F 1,9 =4.37, p=.048).

By contrast, rats in the UNP group did not show interaction effects to indicate reversal of freezing responses to the CX versus CS in the transition from testing to training trials (day 1: F 1,9 =.00003, p=.99; day 2: F 1,9 =1.35, p=.25; day 3: F 1,9 =4.7, p=.04; day 4: F 1,9 =2.52, p=.12). However, the UNP group did show a tendency for greater freezing to the CX than the CS during training sessions on days 2–4 (day 1: F 1,9 <.00001, p=.99; day 2: F 1,9 =5.46, p=.044; day 3: F 1,9 =9.16, p=.014; day 4: F 1,9 =5.05, p=.051). This effect was much smaller in the UNP group than in the PAIR group, and probably occurred for a different reason, as will be explained below.

Transition from freezing to turning responses

To determine why rats behaved differently during testing versus training sessions, we conducted further analyses of their movement behavior. Figure 1B plots the average movement speed of the rats across trials on each day. Since freezing is inversely correlated with movement speed, the graphs in Figure 1B look similar to those in Figure 1A , except that the signs of the measurements are reversed. Movement speeds were analyzed using the same 2×2×2 ANOVA design described above for the freezing analysis. The three-way interaction between stimulus and trial type was again significant on all but the first day of the experiment (day 1: F 1,32 =1.8, p=.19; day 2: F 1,32 =25.7, p=.00002; day 3: F 1,32 =17.4, p=.0002; day 4: F 1,32 =18.0, p=.0002). Newman-Keuls posthoc comparisons showed that after day 1, the PAIR group expressed significantly higher movement speeds during the CS than the CX for training trials (day 1: p=.86; day 2: p=.002; day 3: p=.009; day 4: p=.01). Rats in the UNP group also showed higher movement speeds during the CS than the CX, but this difference was not significant on any day (day 1: p=.34; day 2: p=.26; day 3: p=.17; day 4: p=.25), which is consistent with results reported above showing that both groups froze more to the CX than CS during training sessions, but the effect was more pronounced in the PAIR group. These results indicate that after acquisition of fear conditioning, rats in the both groups froze more and moved less to the CX during training sessions, but the effect was much larger in the PAIR group, and as will be seen below, the underlying cause for this effect was probably different for the PAIR versus UNP groups.

To examine the underlying causes of these conditioned movement responses, we analyzed the rats' turning behavior by using the video tracking system to compute the angular velocity (in degrees/s) of the rat's head throughout each experimental trial (see Methods). This analysis revealed that rats in the PAIR group (but not the UNP group) expressed turning responses to the CS during training trials. These turning responses were strongly biased in the direction opposite from the eyelid where shock was anticipated, as would be expected if they were a flight response away from the shock. Figure 1C plots the average angular turning velocity across trials on each day. Turning responses were analyzed using the same 2×2×2 ANOVA design described above for the freezing and movement analyses. Once again, the three-way interaction between stimulus and trial type was significant on every day after the first conditioning day (day 1: F 1,32 =2.6, p=.12; day 2: F 1,32 =5.1, p=.03; day 3: F 1,32 =15.8, p=.004; day 4: F 1,32 =16.3, p=.003). Newman-Keuls posthoc comparisons revealed that during test trials, rats in the PAIR group showed no significant difference in turning behavior during the CX versus CS on any day (day 1: p=1.0; day 2: p=.99; day 3: p=.95; day 4: p=.96). But during training trials, the PAIR group showed a bias for turning away from the shocked eyelid during the CS (day 1: p=.002; day 2: p=.008; day 3: p=.0001; day 4: p=.0001). It is clear in Figure 1C that this turning bias was absent during test trials at the beginning of each day, and then emerged during the first few training trials and persisted throughout the rest of the day's training trials. By contrast, rats in the unpaired group never showed a significant turning bias to the CS versus CX during test trials (day 1: p=.83; day 2: p=.95; day 3: p=.83; day 4: p=.99) or training trials (day 1: p=.67; day 2: p=.38; day 3: p=.93; day 4: p=.82) on any day of the experiment.

These results indicate that during training sessions, rats in the PAIR group did not freeze less to the CS than CX because they were less afraid during the CS than the CX, but because they expressed their fear of the CS in a different way: by turning rather than freezing. But if this is true, then why did rats in the UNP group also freeze less to the CS than CX, despite showing no turning behavior at all during the CS? Almost certainly, this was because UNP rats actually were less afraid during the CS than the CX. The UNP rats were trained with explicitly unpaired presentations of the CS and US, and it is well established that this procedure can cause the CS to become a “learned safety” signal that inhibits fear responses ( Rescorla, 1969 ). Learned safety effects are not normally measured during training sessions, because freezing behavior tends to be dominated by responses to the US during training ( Rogan et al., 2005 ). This may account for why inhibition of freezing by the CS during training trials was so weak in the UNP group. Based on prior evidence, inhibition of CX-evoked freezing by the explicitly unpaired CS should have been easier to observe when the CS was presented to UNP rats that had not recently encountered the US, during test trials. But the learned safety effect was absent during test trials in the UNP group, because there was very little CX-evoked freezing for the CS to inhibit during these trials. A likely explanation for this is that in hungry UNP rats that had not recently been shocked, the pellet-chasing task caused movements that occluded the CX-evoked freezing responses that would otherwise have been expressed during test trials.

Induction of turning by the US alone

Turning behavior in the PAIR group was not observed during test trials at the beginning of each day, but emerged only during training trials after delivery of the shock US had resumed on that day. To test whether this transition could be induced by the US alone (without the CS), trained rats (n=12) were given 8 presentations of the US alone prior to a test session at the beginning of the day (the first CS alone trial was given 120–240 s after the last US alone trial, the standard intertrial interval). The US alone was delivered either to trained or untrained eyelid in counterbalanced order on different days, so that all rats received the US alone on each eyelid.

Figure 2A shows that when rats were not shocked prior to the test session, they exhibited no turning behavior during the CS alone trials, consistent with data from days 1–4 of the acquisition experiment (see Figure 1C ). Confirming this, a 2×6 ANOVA of turning responses revealed no main effect of stimulus (CX vs. CS repeated: F 1,55 =0.17, p=.69) or trial (1–6 repeated: F 5,55 =0.49, p=.78), and no stimulus-by-trial interaction (F 5,55 =0.54, p=.75). Figure 2B shows that when rats received the US alone to the trained eyelid prior to the test session, they exhibited turning responses during the first few CS alone trials, which diminished over the course of the test session. Confirming this, a 2×6 ANOVA revealed a significant main effect of stimulus (CX vs. CS repeated: F 1,55 =12.97, p=.004) but not trial (1–6 repeated: F 5,55 =1.47, p=.21), and a significant stimulus-by-trial interaction (F 5,55 =3.08, p=.016). Figure 2C shows that when rats received the US alone to the untrained eyelid prior to the test session, they behaved exactly as when they had been shocked on the trained eyelid: that is, they exhibit turning responses during the first few CS alone trials, which diminished over the course of the test session. Confirming this, a 2×6 ANOVA revealed a significant main effect of stimulus (CX vs. CS repeated: F 1,55 =4.85, p=.049) but not trial (1–6 repeated: F 5,55 =1.79, p=.12), and a significant stimulus-by-trial interaction (F 5,55 =2.84, p=.024). Importantly, the turning responses observed after the US alone were always directed away from the trained eyelid, even when the unpaired US had been delivered to the untrained eyelid. Hence, the directional orientation of the turning response depended upon which eyelid had received the US during training by CS-US pairings, and not upon which eyelid the unpaired US had recently been delivered to. This implies that the recent shock did not affect the previously learned association between the CS and US, but instead served as a trigger for changing the rat's defensive responding from a purely passive mode (freezing) to a more active mode that included turning behavior.

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Each graph shows mean CS-evoked turning scores over 6 trials of a test session (n=12 rats). A) Standard test session that was not preceded by shocks. B) Test session preceded by 8 unpaired shocks to the trained side. C) Test session preceded by 8 unpaired shocks to the untrained side.

Experiment 2: Dependence of freezing and turning upon amygdala and PAG

Cannula placements.

The PAIR group in Figure 1 consisted of 24 rats, and 14 of these (AMG group) were implanted with amygdala infusion cannula, while 10 (PAG group) were implanted with PAG infusion cannula. In two AMG rats, the periorbital stimulus wires stopped functioning (that is, they ceased delivering current) prior to completion of the infusion series, so these rats were dropped from the infusion experiments, reducing the size of the AMG group to n=12. Cannula tip placements for the AMG and PAG groups are shown in Figures 3A and 3B , respectively.

Drug diffusion

Figure 3C shows imaging of fluorescent muscimol infused into two rats (which were not included in the behavioral study, see Methods) at the targeted injection sites in AMG and PAG, to illustrate the extent of drug diffusion away from the injection sites at the concentration (0.25 μg/μl) and volume (0.4 μl) used in our experiments. In the AMG group, injectors were targeted at the amygdala's lateral nucleus, but Figure 3A shows that some placements were near enough to the borders of the basal or central nuclei that the drug may have diffused into these areas as well. In the PAG group, cannula placements were concentrated mainly in the lateral and ventral columns ( Figure 3B ), but back-diffusion of the drug along the cannula track may have inactivated neurons in the dorsal column as well ( Figure 3C shows diffusion away from an injector tip in the lateral column). It is also possible that muscimol may have affected the overlying superior colliculus, which is known to play a role in oriented movements, and this could in part account for some of the effects reported below upon turning behavior after PAG infusions. We observed pronounced hemispheric effects following unilateral PAG infusions (see below), so unilateral infusions apparently did not spread significantly into the opposite hemisphere. We have recently shown that acquisition of conditioned freezing is blocked by pre-training infusions of muscimol into PAG—but not into lateral offsite control locations—using the same coordinates, concentration, and volume of the drug as in the present study ( Johansen et al., 2010 ). These prior findings support the likelihood that drug effects observed here were probably not caused by lateral diffusion of muscimol out of PAG into other brain regions.

Experimental procedures

After the rats had been trained over 4–7 days of CS-US pairings (see Methods), they were given muscimol infusions to inactivate their respectively implanted brain regions. Each rat in the AMG and PAG groups received a unilateral infusion of muscimol (0.4 μl per side, 0.25 μg/μl) into the hemisphere ipsilateral or contralateral from the shocked eyelid (counterbalanced over rats), followed by a standard experimental session of 6 test trials and 16 training trials. The experiment was then suspended for a three-day recovery period, followed by a drug-free retraining session on the fourth day after the infusion. On the day after retraining, a second unilateral infusion was given into the hemisphere opposite from the prior infusion. The experiment was then suspended for another three-day recovery period, followed by another drug-free retraining session on the fourth day. On the next day, the PAG group received bilateral muscimol infusions, but the AMG group did not (instead, the AMG group underwent testing for the effects of unpaired shock delivery upon turning behavior, with results reported above in Figure 2 ). Using this repeated measures design, each rat in the AMG and PAG groups received separate unilateral infusions of muscimol into each hemisphere (ipsilateral and contralateral from the trained eyelid), and the PAG group received bilateral infusions as well. We did not examine the effects of bilateral amygdala infusions in the present study.

The effects of muscimol infusions upon freezing and turning behavior were analyzed by performing 2×2 ANOVAs on behavior scores with stimulus (CX vs. CS) and inactivation (pre vs. post) both as repeated factors. Separate ANOVAs were performed to analyze the effects of infusions into each hemisphere (ipsilateral, contralateral, and bilateral). In all analyses presented below, “ipsilateral” and “contralateral” infusion hemispheres are defined with respect to the trained eyelid for each individual rat (for example, ipsilateral infusions were in the left hemisphere for rats trained on the left eyelid, and the right hemisphere for rats trained on the right eyelid, and vice versa for contralateral infusions). For each ANOVA, the “pre” level of the inactivation factor was always comprised from scores obtained during the experimental session that was conducted on the drug-free day immediately prior to the inactivation analyzed by that ANOVA. All posthoc comparisons were performed using the Newman-Keuls test.

Effects of amygdala inactivation on conditioned freezing responses

The effects of infusions upon conditioned freezing behavior were assessed by analyzing behavioral data only from test (CS alone) trials, since CS-evoked freezing was most prominent during these trials (see Figure 1A ). Figure 4A summarizes the effects of amygdala inactivation upon freezing behavior during the CX and CS periods of the test trials. The left graph shows that freezing behavior was not affected by inactivation of the amygdala ipsilateral to the US. Confirming this, the 2×2 ANOVA revealed a main effect of stimulus (F 1,11 =14.67, p=.003) indicating that rats generally froze more to the CS than CX, and a posthoc comparison detected no significant change in CS-evoked freezing after the infusion (p=.61). By contrast, the right graph in Figure 4A shows that contralateral amygdala inactivation significantly reduced CS-evoked freezing responses. The 2×2 ANOVA still exhibited a main effect of stimulus (F 1,11 =9.96, p=.009) because of CS-evoked freezing prior to the infusion, but a posthoc comparison revealed that freezing to the CS was significantly reduced after the infusion (p=.036). This pattern of results replicates our findings from a previous study, in which it was shown that when rats were trained to fear an auditory CS by pairing it with a unilateral US (as in the present study), expression of CS-evoked freezing was impaired by pre-test inactivation of the amygdala contralateral but not ipsilateral from the US ( Blair et al., 2005a ).

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Each graph shows averaged freezing scores before (PRE) versus after (POST) muscimol infusions into a particular hemisphere (IPSI, CONTRA, or BILAT) of the amygdala (panel A) or PAG (panel B). Symbols above black bars denote significance of the comparison with the adjacent white bar. Symbols above connector lines denote significance of the comparison between bars connected by that line.

Effects of PAG inactivation on conditioned freezing responses

Figure 4B summarizes the effects of PAG inactivation upon freezing behavior during the CX and CS periods of the test trials. The leftmost graph shows that ipsilateral PAG inactivation did not significantly affect freezing. The 2×2 ANOVA revealed a main effect of stimulus (F 1,9 =13.7, p=.005) indicating that freezing responses were evoked by the CS, and although CS-evoked freezing was slightly reduced after ipsilateral PAG inactivation, this reduction did not reach significance in a posthoc comparison (p=.12). The middle graph of Figure 4B shows that after contralateral PAG inactivation, freezing levels tended to increase. There was once again a main effect of stimulus (F 1,9 =52.8, p=.00005) to indicate the presence of CS-evoked freezing, but the main effect of drug also approached significance (F 1,9 =4.16, p=.07), and posthoc comparisons revealed significantly increased freezing to both the CX (p=.0003) and CS (p=.009). The rightmost graph in Figure 4B shows that CS-evoked freezing was reduced after bilateral PAG inactivation. Once again there was a main effect of stimulus (F 1,9 =14.8, p=.004) to indicate the presence of CS-evoked freezing before inactivation, and CS-evoked freezing was reduced after the infusion (p=.057); although this reduction in freezing did not quite reach the .05 level for statistical significance, it is consistent with many prior studies showing that disruptions of PAG impair the expression of freezing behavior ( Liebman et al., 1970 ; LeDoux et al., 1988 ; Borszcz et al., 1989 ; Fanselow, 1991 ; Kim et al., 1993 ; Johansen et al., 2010 ).

Effects of amygdala inactivation on conditioned turning responses

Figure 5A summarizes the effects of amygdala inactivation upon turning responses. The left graph shows that ipsilateral amygdala inactivation had no effect upon the rats' turning responses. The 2×2 ANOVA revealed a main effect of stimulus (F 1,11 =25.0, p=.0004), reflecting that fact that rats turned away from the trained eyelid during the CS but not during the CX. Posthoc comparisons showed that this CS-evoked turning bias was present both before (p=.0006) and after (p=.001) inactivation of the ipsilateral amygdala. There was no main effect of drug (F 1,11 =1.71, p=.22) and no stimulus-by-drug interaction (F 1,11 =0.27, p=.61). Although CS-evoked turning was reduced slightly after the infusion, this reduction was not significant (p=.24), and turning behavior during the CX also remained unchanged (p=.63).

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Each graph shows averaged turning scores before (PRE) versus after (POST) muscimol infusions into a particular hemisphere (IPSI, CONTRA, or BILAT) of the amygdala (panel A) or PAG (panels B and C). Symbols adjacent to connector lines denote significance of the comparison between dots connected by that line.

The right graph shows that contralateral amygdala inactivation completely abolished CS-evoked turning behavior. Confirming this, the 2×2 ANOVA revealed a main effect of both stimulus (F 1,11 =10.1, p=.009) and drug (F 1,11 =12.6, p=.005), as well as a stimulus-by-drug interaction (F 1,11 =12.3, p=.005). CS-evoked turning was dramatically reduced after the infusion (p=.003), while turning behavior during the CX (which was absent to begin with) remained unchanged (p=.9). Posthoc comparisons also showed that rats turned away from the trained eyelid significantly more during the CS than the CX before inactivation (p=.003), but not after the inactivation (p=.89). These results indicate that much like conditioned freezing responses, conditioned turning responses also appear to depend preferentially upon the amygdala contralateral from the trained eyelid in our fear conditioning task.

Effects of PAG inactivation on conditioned turning responses

Figure 5B summarizes the effects of PAG inactivation upon turning behavior. The leftmost graph shows that ipsilateral PAG inactivation significantly altered the rats' turning responses. The 2×2 ANOVA revealed a main effect of stimulus (F 1,9 =12.0, p=.007), reflecting that fact that rats turned away from the trained eyelid significantly more during the CS than the CX. Although posthoc comparisons showed that the CS-evoked turning bias was present both before (p=.003) and after (p=.02) inactivation of the ipsilateral PAG, there was also a main effect of drug (F 1,9 =7.33, p=.02) with no stimulus-by-drug interaction (F 1,9 =0.86, p=.38). These effects reflected the fact that compared to the pre-inactivation baseline, ipsilateral PAG inactivation caused turning responses to shift toward the trained eyelid by a similar amount during both the CX (p=.04) and CS (p=.005).

The middle graph in Figure 5B shows that contralateral PAG inactivation also altered the rats' turning behavior, but in a different way from ipsilateral PAG inactivation. The main effects of stimulus (F 1,9 =3.55, p=.09) and drug (F 1,9 =4.31, p=.07) no longer reached significance (although both showed trends at p<.1), whereas the interaction between stimulus and drug was highly significant (F 1,9 =31.54, p=.0003). This pattern reflected the fact that rats once again turned away from the trained eyelid significantly more during the CS than the CX before inactivation (p=.001), but after inactivation the direction of the turning bias was reversed, so that rats now showed a trend to turn away from the trained eyelid more during the CX than the CS (p=.06). This reversal occurred because contralateral PAG inactivation caused the rats to stop turning during the CS (p=.001; this may also be regarded as a shift of the turning direction toward the trained eyelid), while at the same time causing a trend for the turning direction to shift away from the trained eyelid during the CX (p=.06), despite a baseline of no CX turning at all prior to inactivation.

The rightmost graph in Figure 5B shows that the effects of bilateral PAG inactivation were similar to the effects of contralateral inactivation. The main effects of stimulus (F 1,9 =1.11, p=.32) and drug (F 1,9 =1.27, p=.29) were not significant, but the interaction between stimulus and drug was significant (F 1,9 =18.99, p=.002). Rats turned away from the trained eyelid significantly more during the CS than the CX before inactivation (p=.006), but this turning bias was eliminated after inactivation. Bilateral PAG inactivation abolished turning behavior during the CS (p=.006) while having no effect on turning during the CX (p=.16), which was absent to begin with. After bilateral PAG inactivation, rats showed no significant difference in turning responses to the CX versus CS (p=.18).

Persistent turning after unilateral PAG inactivation

Unilateral PAG inactivation induced persistent turning during the CX period of training sessions, which was always directed toward the inactivated hemisphere (that is, towards the side of US delivery for IPSI inactivations, and away from the side of US delivery during CONTRA inactivations). To further investigate this, we analyzed turning responses during test trials that were given after PAG inactivation but before the US had been delivered.

Figure 5C summarizes the effects of PAG inactivation upon turning responses during test sessions. Uninfused rats (“PRE”) never exhibited turning responses to the CX or CS during test trials, in agreement with findings reported above (see Figure 1C ). The leftmost graph in Figure 5C shows that ipsilateral PAG inactivation induced persistent turning toward the side of the infusion (or equivalently, toward the trained eyelid) during both the CX and CS. Confirming this, the 2×2 ANOVA revealed no main effect of stimulus (F 1,9 =1.09, p=.32) but a significant main effect of drug (F 1,9 =19.0, p=.002), with posthoc comparisons indicating a shift of turning responses toward the inactivated hemisphere during both the CX (p=.02) and CS (p=.001). The middle graph in Figure 5C shows that contralateral PAG inactivation also induced persistent turning, but this time in the direction away from the trained eyelid (which was once again toward the inactivated hemisphere, as in the case of ipsilateral inactivation). Confirming this, the 2×2 ANOVA revealed no main effect of stimulus (F 1,9 =0.76, p=.4) but a significant main effect of drug (F 1,9 =10.6, p=.01), and posthoc comparisons revealed that PAG inactivation caused turning responses to shift toward the inactivated hemisphere during both the CX (p=.002) and CS (p=.001). The right most graph in Figure 5C shows that unlike unilateral inactivations, bilateral PAG inactivation did not induce persistent turning during test sessions. Confirming this, the 2×2 ANOVA revealed no main effect of stimulus (F 1,9 =0.84, p=.38) or drug (F 1,9 =0.29, p=.6) for bilateral PAG inactivation.

In sum, our findings indicate that unilateral (but not bilateral) inactivation of PAG induced persistent turning toward the inactivated hemisphere. This agrees with prior data showing that unilateral inhibition of PAG and surrounding areas can produce turning oriented toward the inactivated side ( Geula and Asdourian, 1984 ). This drug-induced turning behavior complicates our ability to interpret the effects of PAG inactivation upon CS-evoked turning responses, but we shall argue in the Discussion section that the pattern of results observed here is consistent with the possibility that PAG plays a primary role in the expression of CS-evoked turning responses following post-encounter potentiation of conditioned fear.

Effects of muscimol on US processing

Figure 5 showed that conditioned turning responses could be impaired by inactivation of either the amygdala or PAG, especially on the side contralateral (but not ipsilateral) from the trained eyelid. There are two possible ways that inactivation might have produced this impairment of turning behavior. First, inactivation may have shut down neural pathways through the amygdala and PAG that directly mediate expression of CS-evoked turning responses. However, since CS-evoked turning responses do not occur unless rats have recently encountered the US (see Figure 1C ), a second possibility is that inactivation may have interfered with nociceptive pathways that mediate sensory processing of the US, and thereby prevented the US from inducing the turning response. These two explanations are not mutually exclusive, since the amygdala and PAG both participate in defensive behavior as well as nociception ( Bandler and DePaulis, 1991 ; Davis, 1992 ; Keay et al., 1997 ; Jordan, 1998 ; Guariau and Bernard, 2004 ; Neugebauer, 2006 ).

To investigate how US processing was affected by muscimol, we examined unconditioned movement responses evoked by the US before and after inactivations of amygdala and PAG. Figure 6 shows average movement speeds during US delivery before and after muscimol infusions. It was found that inactivation of the amygdala ipsilateral to the trained eyelid did not affect US-evoked head movements ( Figure 6A , left graph), but inactivation of the contralateral amygdala attenuated US-evoked head movements ( Figure 6B , right graph). Confirming this, a 2×2 ANOVA of movements speeds with hemisphere (ipsilateral vs. contralateral) and amygdala inactivation (pre vs. post) as repeated factors yielded main effects of hemisphere (F 1,11 =16.8, p=.002) and inactivation (F 1,11 =38.8, p=.00006), as well as a significant interaction between hemisphere and inactivation (F 1,11 =9.29, p=.01). Posthoc comparisons indicated that the average speed of head movements was unchanged after ipsilateral amygdala inactivation (p=.14), but significantly lower after than before contralateral amygdala inactivation (p=.0007). US-evoked responses after contralateral inactivation were also lower than after (p=.002) or before (p=.0006) ipsilateral inactivation. In a prior study, we showed that the periorbital shock US elicits unconditioned head movements from rats, and that these movements are attenuated by bilateral lesions or inactivation of the amygdala ( Blair et al., 2005b ). Our present findings suggest that the contralateral (but not ipsilateral) amygdala may be the primary contributor to nociceptive processing of the US, which in turn may partly account for the data above showing that the US was no longer able to induce post-encounter potentiation of fear responses after contralateral amygdala inactivation ( Figure 5A , right graph).

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Each graph shows averaged movement speed during the US before (PRE) versus after (POST) muscimol infusions into a particular hemisphere (IPSI, CONTRA, or BILAT) of the amygdala (panel A) or PAG (panel B). Symbols adjacent to connector lines denote significance of the comparison between bars connected by that line.

Figure 6B shows average movement speeds during US delivery before and after muscimol infusions into PAG. Inactivation of the PAG ipsilateral to the trained eyelid did not affect US-evoked head movements ( Figure 6A , left graph), but inactivation of the contralateral PAG attenuated US-evoked head movements ( Figure 6B , middle graph), as did bilateral PAG inactivation ( Figure 6B , right graph). Confirming this, a 3×2 ANOVA of movements speeds with hemisphere (ipsilateral vs. contralateral vs. bilateral) and PAG inactivation (pre vs. post) as repeated factors yielded main effects of hemisphere (F 2,18 =5.9, p=.01) and inactivation (F 1,18 =136.5, p<.00001), but no interaction between hemisphere and inactivation (F 2,18 =2.08, p=.15). Posthoc comparisons indicated that the average speed of head movements was only trending toward reduction after ipsilateral PAG inactivation (p=.16), but significantly reduced after contralateral (p=.0009) or bilateral (p=.001) inactivation of PAG. Hence, it appears that the contralateral (but not ipsilateral) PAG may play a role in nociceptive processing of the US, and this might partially explain why post-encounter potentiation of conditioned fear does not occur after contralateral or bilateral inactivation of PAG.

In studies presented here, rats were trained to fear an auditory CS by pairing it with a mild electric shock US delivered to one eyelid, as in prior studies from our laboratory ( Moita et al. 2003 , 2004 ; Blair et al, 2005a , b ; Tarpley et al., 2009 ; Johansen et al., 2010 ). After training, the CS elicited conditioned freezing responses when rats that had not recently encountered the US, but when the same CS was presented to trained rats that had recently encountered the US, it elicited turning in circles away from the trained eyelid where US delivery was anticipated to occur. The CS never elicited such turning responses from rats that were given unpaired presentations of the CS and US, regardless of whether they had recently encountered the US. Pharmacological inactivation experiments revealed that CS-evoked freezing and turning responses were both dependent upon lateralized circuits in the amygdala and PAG, which are known to mediate acquisition and expression of associative fear conditioning (for review, see Fanselow, 1991 ; Davis, 1992 ; Maren, 1998; LeDoux, 2000 ). These findings indicate that fear conditioned rats can express different defensive responses (residing at different points along the predatory imminence continuum) to the same CS, depending upon their recent history of aversive encounters.

Turning as a post-encounter defensive response

When trained rats had not recently encountered a shock, they were presumably in a pre-encounter state of low predatory imminence. Hence, they engaged in pellet-chasing behavior while the CS was not being presented (this is reflected by high movement and low turning scores during the CX period of test trials for paired rats in Figures 1B and 1C ). It should be noted that the platform CX may have acquired some association with the shock US via background contextual fear conditioning (since the US was always delivered while rats chased food pellets on the platform). But when rats had not recently been shocked, such context conditioning was apparently not strong enough for the CX to elicit reliable freezing or turning responses by overcoming the rats' competing motivation to chase pellets on the platform. Hence, in rats that had not recently been shocked (that is, during test trials), the CX period appeared to be a time of low predatory imminence during which rats engaged in goal-seeking behavior (that is, pellet chasing). When the CS was presented to trained rats that had not recently been shocked (during test trials at the beginning of each day), they exhibited freezing but not turning responses (reflected by high freezing and low turning scores during the CS on test trials for paired rats in Figures 1A and 1C ). Hence, rats that had not recently been shocked may have perceived the CS as a “circa-strike” threat—analogous to a distant predator that has not yet detected the rat's presence—for which the ethologically programmed response was freezing to avoid detection.

When trained rats had recently encountered a shock, their behavior during both the CX and CS became “shifted to the right” along the predatory imminence continuum. Behavior during the CX after the shock resembled that during the CS prior to the shock: high freezing and low turning scores (see CX data from training trials for paired rats in Figures 1A and 1C ). This emergence of CX-evoked freezing could either reflect post-shock freezing elicited by the US, or a latent CX-US association that was too weak to overcome the pellet-chasing drive before shock delivery, but was unmasked after recent encounters with the shock. Consistent with the second possibility, behavior during the CS after the shock was characterized by the emergence of conditioned turning, directed away from the trained eyelid (as indicated by the high turning and moving scores—along with low freezing scores—during training trials for paired rats in Figures 1A–C ). Hence, rats that had recently been shocked may have perceived the CS as a “post-encounter” threat—analogous to a predator that has already detected the rat's presence and recently mounted an attack—for which the ethologically programmed response was flight to avoid/escape further attack. Supporting this interpretation, CS-evoked turning responses exhibited two hallmark characteristics of a post-encounter flight response: 1) turning was expressed preferentially when the CS was presented after a recent encounter with an aversive US (analogous to recent injury or predatory attack), and 2) turning was oriented in the direction away from the site of anticipated US delivery, moving the rat away from the source of danger.

Neural substrates for fear conditioning and defensive behavior

The perception of threat is often triggered by cues in the environment which signal the presence of danger (such as a fear-conditioned CS), and the amygdala is thought to play a key role in attaching motivational valence to such cues, which allows the organism to accurately perceive the present level of threat in its environment ( Weiskrantz, 1956 ; Davis, 1992 ; Davis and Whalen, 2001 ; LeDoux, 2000 ; Blair et al., 2005b ; Seymour & Dolan, 2008 ). Output from the amygdala is relayed to the PAG ( Rizvi et al., 1991 ), which is one of the low-level brain structures that is critically involved in coordinating the performance of defensive behaviors in response to threats ( Chaurand et al., 1972 ; LeDoux et al., 1988 ; Bandler & DePaulis, 1991 ; Fanselow, 1991 ; DePaulis et al., 1992 ; Behbehani, 1995 ; Jordan, 1998 ; Mobbs et al., 2007 , 2009 ). If we regard the predatory imminence continuum as an innate “defense policy” which maps different threat states onto particular strategies for evasive action, then it is reasonable to speculate that the current threat level may be signaled by the amygdala, and then relayed (either directly or indirectly) to the PAG, where it is converted into a defensive response that is appropriate for the current threat. Our present findings are consistent with this point of view, and suggest new avenues for future research to investigate how different levels of threat are encoded and converted into specific behavioral responses by the brain's fear system.

Role of the amygdala in CS versus US processing

It is believed that convergence of sensory information about the CS and US onto single amygdala neurons can trigger Hebbian plasticity at the synapses which relay the CS to those neurons, thereby storing a memory of the CS-US association ( LeDoux et al., 2000 ; Blair et al., 2001; Maren, 2005 ). Supporting this view, it has been shown that the amygdala plays an important role in many conditioned defensive responses, including freezing, potentiated startle, autonomic changes in heart rate and blood pressure, and conditioned analgesia ( LeDoux et al., 1988 ; Davis, 1992 ; Kapp et al., 1992 ; Helmstetter, 1992 ; Choi et al., 2001 ).

In the present study, we observed that two different conditioned fear responses—freezing and turning—were both similarly dependent upon the amygdala ( Figures 4 and and5), 5 ), especially the hemisphere contralateral from the US (see below). This suggests that both conditioned responses depended upon the same memory of the CS-US association, which was stored primarily in one hemisphere of the amygdala. However, since conditioned turning responses were only elicited by the CS after recent encounters with shock, inactivation of the contralateral amygdala may have impaired turning responses not only by blocking recall of the CS-US association, but also by interfering with the ability of the shock to induce turning behavior. Supporting this possibility, we observed here that unilateral inactivation of the amygdala impaired movement responses evoked by a US delivered to the eyelid contralateral from the inactivation ( Figure 6 ). Hence, amygdala inactivation may have impaired CS-evoked turning responses in two different ways: by blocking recall of the CS-US association, and by also attenuating the aversiveness of the shocks upon which turning behavior depended (see Blair et al., 2005b ). A good way to further test the role of the amygdala in mediating associative recall versus shock aversiveness would be to inactivate the amygdala ipsilateral to the trained eyelid (which should not impair associative recall) prior to delivering unpaired shocks to the untrained eyelid (opposite from the inactivated hemisphere). We predict that the CS would only elicit freezing but not turning responses in this case, since the memory for the CS-US association would remain intact in the hemisphere contralateral from the trained eyelid (driving the freezing response), but the unpaired shocks would no longer be able to induce turning because their aversiveness would be attenuated by inactivation of the amygdala contralateral from the shock.

Lateralized processing of aversive stimuli by the amygdala

We have previously shown that disruption of the amygdala contralateral (but not ipsilateral) from an eyelid shock US impairs conditioned freezing responses to a CS that predicts the US ( Blair et al, 2005a ; Tarpley et al., 2009 ). Here, we found that CS-evoked turning responses and US-evoked reflex movements were also dependent upon the amygdala contralateral from the US. Taken together, these findings suggest that aversive stimulation from the left eyelid may be processed by the right amygdala, and vice versa. However, there is also a body of evidence which suggests that aversive stimuli are processed mainly in the right amygdala rather than the left, regardless of their source (LaLumiere and McGaugh, 1995; Canli et al. 1998 ; Funayama et al., 2001 ; Baker and Kim, 2004 ). For example, it has been shown in rodents that a nociceptive stimulus delivered to either side of the body (left or right) activates chemical and physiological responses primarily in the right rather than the left amygdala ( Ji and Neugebauer, 2009 ; Carasquillo and Gereau, 2008 ). But these studies examined amygdala responses to chronic inflammatory pain in the limbs, which is relayed to the amygdala via the anterolateral spinal pathway. By contrast, the eyelid shock US in our studies was an acute aversive stimulus, relayed to the amygdala via trigeminal pathways (see below). Different types of nociceptive stimuli (such as acute versus chronic stimuli, or spinal versus trigeminal stimuli) can elicit a broad variety of different defensive behaviors, and it is possible that lateralization of aversive stimulus processing in the amygdala may depend upon what behaviors are elicited by a particular aversive stimulus.

The turning response we have characterized here is a highly lateralized response, directed toward one side of the body and away from the other. The fact that this turning response depends upon the amygdala contralateral from aversive stimulation (and therefore, ipsiversive to the direction of turning) might provide a clue to the underlying functional reasons why conditioned fear of a unilateral eyelid shock US is processed preferentially in one amygdala hemisphere. Nociceptive signals from the eyelid enter the brain through the ipsilateral trigeminal nucleus of the medullary dorsal horn, which sends weak projections directly to the basal and central nuclei of the contralateral amygdala, and strong (predominantly contralateral) projections to the posterior intralaminar thalamus ( Cliffer et al., 1991 ; Guariau and Bernard, 2004 ), which in turn sends uncrossed projections to several amygdala subnuclei ( LeDoux et al., 1987 ; 1990 ). Projections from the central nucleus of the amygdala to PAG are ipsilaterally biased ( Rizvi et al., 1991 ), so outputs from each amygdala hemisphere may preferentially be relayed to PAG on the same side of the brain to drive defensive responses in the proper direction (see below).

Role of the PAG in mediating defensive responses

Disruption of PAG has been shown to impair a variety of defensive responses to threatening stimuli ( Liebman et al., 1970 ; LeDoux et al., 1988 ; Borszcz et al., 1989 ; Fanselow, 1991 ; Kim et al., 1993 ; Helmstetter & Tershner, 1994 ; Johansen et al., 2010 ). In addition, electrical or pharmacological stimulation of PAG can elicit “fight-or-flight” behaviors in the absence of any threatening stimulus, indicating that neural activity in PAG is sufficient for expressing defensive behaviors ( DiScala et al., 1984 ; Bandler & DePaulis, 1991 ; DePaulis et al., 1992 ; Keay & Bandler, 2001 ). Recent evidence from human imaging studies shows that blood oxygen levels in PAG are increased during close encounters with threatening stimuli, suggesting that metabolic activity in PAG is correlated with post-encounter defensive responding ( Mobbs et al., 2007 , 2009 ). These findings support the view that expression of conditioned fear responses may depend upon projections from the amygdala, where memories of the CS-US association are stored, to the PAG, which reads out these memories to drive the expression of various conditioned defensive behaviors ( LeDoux et al., 1988 ; Fanselow, 1991 ; 1994 ; Zhao and Davis, 2004 ). It has also been proposed that PAG might be an additional site of associative plasticity (outside of the amygdala) where components of the CS-US association might be stored ( Helmstetter et al., 2008 ). Evidence suggests that the ventral PAG (vPAG) drives passive defensive behaviors such as freezing, whereas the dorsal PAG (dPAG) drives active defensive behaviors, such as flight ( Depaulis et al., 1992 ; De Oca et al., 1998 ; Kim et al., 1993 ; Leman et al., 2003 ; Vianna et al., 2001 ; Fanselow, 1991 ). In the present experiments, muscimol infusions were targeted non-specifically at both the lateral and ventral columns of PAG (see Figure 3B ), but upward diffusion along the cannula tracks probably led to inactivation of overlying dPAG as well, and possibly the superior colliculus ( Figure 3C ). PAG inactivation impaired both freezing and turning responses, as would be expected after inactivation of both the vPAG and dPAG.

Lateralized control of defensive responding by PAG

In our experiments, unilateral inactivation of PAG induced persistent turning behavior towards the inactivated hemisphere ( Figure 5 ), and this finding is consistent with prior evidence showing that ipsiversive turning can be induced by unilateral PAG inactivation ( Geula and Asdourian, 1984 ), as well as evidence for the opposite effect that unilateral excitation of PAG can produce contraversive turning behavior ( DePaulis et al., 1992 ). Based on this pattern of results, it is tempting to conclude that the balance of excitation within the two PAG hemispheres might play a role in “steering” defensive responses in the proper direction to move the animal away from the source of danger. Thus, when the rat experiences or expects delivery of an aversive US on one side of the body, the amygdala may become activated in the contralateral hemisphere, and send outputs to PAG that suppress activity in that hemisphere. This may produce turning behavior towards the side of the activated amygdala and suppressed PAG, and thus, away from the site of US delivery. The superior colliculus overlying PAG may also help to determine the direction of the flight response, since this area is interconnected with PAG ( Mantyh, 1982 ) and plays a role in determining the directional orientation of defensive behaviors ( Geula and Asdourian, 1984 ).

Inactivation of PAG contralateral to the trained eyelid impaired turning ( Figure 5B ), but spared and may even have enhanced freezing ( Figure 4B ), perhaps by impairing the competing turning response. Inactivation of PAG ipsilateral to the trained eyelid also spared freezing, but bilateral PAG inactivations impaired freezing ( Figure 4B ), implying that our muscimol infusions did affect vPAG columns involved in freezing. The tendency for bilateral but not unilateral PAG infusions to impair freezing suggests that both PAG hemispheres may have contributed to the freezing response, even though freezing depends preferentially upon the contralateral amygdala in our task ( Figure 4A ; Blair et al., 2005a ).

Inactivation of PAG ipsilateral to the trained eyelid had an indeterminate effect on turning, because of persistent turning toward the trained eyelid that was induced by the infusion. Even though CS-evoked turning behavior appeared to be absent during training sessions after ipsilateral PAG inactivation ( Figure 5B , leftmost graph), this may not have been the case. Instead, CS-evoked turning away from the trained eyelid may have been spared after the inactivation, but masked (or cancelled out) by persistent turning in the opposing direction, resulting in almost no net CS-evoked turning behavior. That is, the rats may have been struggling during the CS to turn “upstream” against the persistent turning in the wrong direction, and succeeded only in a cessation of turning, rather than a full reversal of the turning direction. However, the lack of CS-evoked turning after ipsilateral PAG inactivation could also have been caused by freezing responses that interrupted persistent turning behavior during the CS but not the CX. The effects of ipsilateral PAG inactivation upon turning behavior were thus difficult to ascertain, but the impairment of turning after contralateral PAG inactivation was unambiguous, and the overall pattern of results is consistent with the interpretation that PAG participates in expression of turning behaviors, in agreement with existing evidence that the PAG plays a role in such behaviors ( Depaulis et al., 1992 ; De Oca et al., 1998 ; Kim et al., 1993 ; Leman et al., 2003 ; Vianna et al., 2001 ; Fanselow, 1991 ).

Possible mechanisms for US-triggered transition from passive to active defense

One possible neurobiological mechanism for the US-induced transition from freezing to turning behavior could be a change in the responsiveness of vPAG and dPAG to inputs from the amygdala. When rats have not recently been shocked, CS-evoked activity in the amygdala may trigger vPAG neurons to produce freezing responses, whereas after rats have recently been shocked, the same CS-evoked activity in the amygdala may instead trigger dPAG neurons to produce flight responses. Perhaps vPAG and dPAG might compete with one another for control over defensive responding (see Walker et al., 1997 ), and shock delivery might alter the balance of this competitive interaction so that prior to shock delivery, competition is biased in favor of the vPAG, but after shock delivery, the bias shifts to favor dPAG. It has been proposed (see Deakin & Graeff, 1991 ) that defensive behavior might be influenced by neuromodulatory systems which are known to regulate neural activity in PAG (such as opiates, dopamine, and serotonin), and activation of these neuromodulators by shocks is one possible mechanism by which the responsiveness of vPAG and dPAG to input from the amygdala might be altered to produce changes in defensive behavior.

In addition to the amygdala-PAG pathway, there is also evidence that the medial prefrontal cortex (mPFC) might play a significant role in the expression of conditioned freezing responses ( Blum et al., 2006 ; Corcoran and Quirk, 2007 ; Burgos-Robles et al., 2009 ). The mPFC receives input from the amygdala and sends major projections to PAG ( McDonald, 1991 ; Floyd et al., 2000 ), so modulation of neural activity in mPFC by shocks could be another mechanism by which shock delivery might alter the rat's defensive response strategy. Further studies are warranted to investigate how different brain structures (including amygdala, mPFC, vPAG, and dPAG) and neuromodulators regulate US-triggered transitions between conditioned freezing and turning responses. It will also be important for future studies to examine whether induction of CS-evoked turning by the US can occur in contexts other than the one where the CS-US association was learned, and whether arousing stimuli other than eyelid shocks can induce CS-evoked turning behavior in fear conditioned rats.

Implications for clinical anxiety disorders

The neural circuits that regulate fear expression have been widely studied, and rodent fear conditioning has been adopted as a dominant animal model for investigating the neurobiological basis of clinical anxiety disorders ( Davis and Whalen, 2001 ; Rau et al., 2005 ; Davis et al., 2006 ; Milad et al. 2006 ; Miller & McEwen, 2006 ; Rauch et al., 2006 ). It has been proposed that patients with anxiety disorders may suffer from over-acquired fear associations, so that neutral cues which should not predict danger (or only predict a moderate level of danger) instead predict a high level of danger, leading to over-activation of the amygdala and inappropriate defense responses ( Davis and Whalen, 2001 ; Rau et al., 2005 ; Rauch et al., 2006 ). In human patients, such inflated fear responses might result from an inability to normally extinguish conditioned fear associations, and this possibility has generated considerable interest in therapies that focus on helping patients with anxiety disorders to extinguish their fear memories ( Davis et al., 2006 ; Milad et al., 2006 ).

However, the findings reported here emphasize that defensive responses depend not only upon long-lasting associative fear memories that have been acquired in the remote past, but also upon transient encounters with aversive stimuli that have occurred in the recent past. In healthy people, a recent brush with danger may acutely enhance defensive responses to mildly threatening stimuli (to promote escape from recently encountered threats that may still be present in the environment), without causing long-term changes in the aversive valence of such stimuli. But in some patients with anxiety disorders, this normally acute state might become chronic, so that the patient always behaves as if they have recently had a brush with danger (even when they have not), and thus always exhibits exaggerated defensive responding to mildly threatening stimuli. In other words, such patients might always behave as if they are in a “training session” (behaving as though a US has recently been delivered) even when they are actually in a “test session” (because no US has in fact been recently encountered).

Our present findings imply that in rodents, the mode of conditioned fear expression depends strongly upon recent (rather than remote) events (see also Mongeau et al., 2003 ). This fact highlights the possibility that neurobiological mechanisms underlying chronically heightened defensiveness might be independent from the long-term memory processes that mediate the storage of CS-US associations. Instead, chronically heightened defensiveness might sometimes be caused by dysfunction of short-term memory systems that register the recent occurrence of aversive events, or by chronic activation of hormonal and neuromodulatory systems that are acutely activated by such aversive events in healthy people. To further investigate how these factors might contribute to anxiety disorders, it will be necessary to identify the neural mechanisms by which encounters with aversive events can acutely modulate the mode of defensive responding. The novel conditioned turning response we have described here may provide a helpful tool for observing and measurinig US-triggered transitions from circa-strike to post-encounter defensive responding in fear conditioned rats, and may thus provide a useful animal model for investigating how brain systems that are acutely activated during aversive stimulus encounters might contribute to clinical anxiety disorders.

Acknowledgements

We thank Michael Fanselow, Bernard Balleine, Josh Johansen, and Adam Welday for helpful comments and discussion. This work was supported by NIH R01 MH073700 and a NARSAD Young Investigator Award to H.T.B.

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Baker KB, Kim JJ. Amygdalar lateralization in fear conditioning: evidence for greater involvement of the right amygdala. Behav Neurosci. 2004; 118 :15–23. [ PubMed ] [ Google Scholar ]
Bandler R, Depaulis A. Midbrain periaqueductal gray control of defensive behavior in the cat and the rat. In: Depaulis A, Bandler R, editors. The midbrain periaqueductal gray matter: Functional, anatomical and neurochemical organization. Plenum Press; New York: 1991. pp. 175–198. [ Google Scholar ]
Behbehani MM. Functional characteristics of the midbrain periaqueductal gray. Prog Neurobio. 1995; 46 :575–605. [ PubMed ] [ Google Scholar ]
Blair HT, Huynh VK, Vaz VT, Van J, Patel RR, Hiteshi AK, Lee JE, Tarpley JW. Unilateral storage of fear memories by the amygdala. J Neurosci. 2005a; 25 :4198–4205. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Blair HT, Sotres-Bayon F, Moita MA, Ledoux JE. The lateral amygdala processes the value of conditioned and unconditioned aversive stimuli. Neuroscience. 2005b; 133 :561–569. [ PubMed ] [ Google Scholar ]
Blanchard DC, Blanchard RJ. Crouching as an index of fear. J Comp Physiol Psychol. 1969a; 67 :370–375. [ PubMed ] [ Google Scholar ]
Blanchard DC, Blanchard RJ. Passive and active reactions to fear-eliciting stimuli. J Comp Physiol Psychol. 1969b; 68 :129–135. [ PubMed ] [ Google Scholar ]
Blum S, Hebert AE, Dash PK. A role for the prefrontal cortex in recall of recent and remote memories. Neuroreport. 2006; 17 :341–344. [ PubMed ] [ Google Scholar ]
Bolles RC. Species-specific defense reactions and avoidance learning. Psychol Rev. 1970; 77 :32–48. [ Google Scholar ]
Borszcz GS, Cranney J, Leaton RN. Influence of long-term sensitization on long-term habituation of the acoustic startle response in rats: central gray lesions, preexposure, and extinction. J Exp Psychol Anim Behav Process. 1989; 15 :54–64. [ PubMed ] [ Google Scholar ]
Burgos-Robles A, Vidal-Gonzales I, Quirk GJ. Sustained conditioned responses in prelimbic prefrontal neurons are correlated with fear expression and extinction failure. J Neurosci. 2009; 29 :8474–8482. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Canli T, Desmond JE, Zhao Z, Glover G, Gabrieli JD. Hemispheric asymmetry for emotional stimuli detected with fMRI. Neuroreport. 1998; 9 :3233–3239. [ PubMed ] [ Google Scholar ]
Carasquillo Y, Gereau RW. Hemispheric lateralization of a molecular signal for pain modulation in the amygdala. Mol Pain. 2008; 4 :24. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Chaurand JP, Vergnes M, Karli P. Substance grise centrale du mésencephale et comportement d'aggression interspécifique du rat. Physiol Behav. 1972; 9 :475–481. [ PubMed ] [ Google Scholar ]
Choi JS, Lindquist DH, Brown TH. Amygdala lesions block conditioned enhancement of the early component of the rat eyeblink reflex. Behav Neurosci. 2001; 115 :764–775. [ PubMed ] [ Google Scholar ]
Cliffer KD, Burstein R, Giesler GJ., Jr Distributions of spinothalamic, spinohypothalamic, and spinotelencephalic fibers revealed by anterograde transport of PHA-L in rats. J Neurosci. 1991; 11 :852–868. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Corcoran KA, Quirk GJ. Activity in prelimbic cortex is necessary for the expression of learned, but not innate, fears. J Neurosci. 2007; 27 :840–844. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Craske MG. Anxiety Disorders: Psychological Approaches to Theory and Treatment. Westview; Boulder, CO: 1999. [ Google Scholar ]
Davis M. The role of the amygdala in fear and anxiety. Annu Rev Neurosci. 1992; 15 :353–375. [ PubMed ] [ Google Scholar ]
Davis M, Myers KM, Chhatwal J, Ressler KJ. Pharmacological treatments that facilitate extinction of fear: relevance to psychotherapy. NeuroRx. 2006; 3 :82–96. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Davis M, Whalen PJ. The amygdala: Vigilance and emotion. Mol Psychiatry. 2001; 6 :13–34. [ PubMed ] [ Google Scholar ]
De Oca BM, DeCola JP, Maren S, Fanselow MS. Distinct regions of the periaqueductal gray are involved in the acquisition and expression of defensive responses. J Neurosci. 1998; 18 :3426–3432. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Deakin JF, Graeff FG. 5-HT and mechanisms of defence. J Psychopharm. 1991; 5 :305–315. [ PubMed ] [ Google Scholar ]
Depaulis A, Keay KA, Bandler R. Longitudinal neuronal organization of defensive reactions in the midbrain periaqueductal gray region of the rat. Exp Brain Res. 1992; 90 :307–318. [ PubMed ] [ Google Scholar ]
Di Scala G, Schmitt P, Karli P. Flight induced by infusion of bicuculline methiodide into periventricular structures. Brain Res. 1984; 309 :199–208. [ PubMed ] [ Google Scholar ]
Fanselow MS. The Midbrain Periaqueductal Gray as a Coordinator of Action in Response to Fear and Anxiety. In: Depaulis A, Bandler R, editors. The midbrain periaqueductal gray matter: Functional, anatomical and neurochemical organization. Plenum Press; New York: 1991. pp. 151–175. [ Google Scholar ]
Fanselow MS. Neural organization of the defensive behavior system responsible for fear. Psychonomic Bull Rev. 1994; 1 :429–438. [ PubMed ] [ Google Scholar ]
Fanselow MS, Lester LS. A functional behavioristic approach to aversively motivated behavior: Predatory imminence as a determinant of the topography of defensive behavior. In: Bolles RC, Beecher MD, editors. Evolution and Learning. Erlebaum; Hillsdale, NJ: 1988. pp. 185–211. [ Google Scholar ]
Floyd NS, Price JL, Ferry AT, Keay KA, Bandler R. Orbitomedial prefrontal cortical projections to distinct longitudinal columns of the periaqueductal gray in the rat. J Comp Neurol. 2000; 422 :556–578. [ PubMed ] [ Google Scholar ]
Funayama ES, Grillon C, Davis M, Phelps EA. A double-dissociation in the affective modulation of startle in humans: effects of unilateral temporal lobectomy. J Cogn Neurosci. 2001; 13 :721–729. [ PubMed ] [ Google Scholar ]
Geula C, Asdourian D. Circling and bodily asymmetry induced by injection of GABA agonists and antagonists into the superior colliculus. Pharmacol Biochem Behav. 1984; 6 :853–858. [ PubMed ] [ Google Scholar ]
Guariau C, Bernard JF. A comparative reappraisal of projections from the superficial laminae of the dorsal horn in the rat: The forebrain. J Comp Neurol. 2004; 468 :24–56. [ PubMed ] [ Google Scholar ]
Helmstetter FJ. The amygdala is essential for the expression of conditional hypoalgesia. Behav Neurosci. 1992; 106 :518–528. [ PubMed ] [ Google Scholar ]
Helmstetter FJ, Parsons RG, Gafford GM. Macromolecular synthesis, distributed synaptic plasticity, and fear conditioning. Neurobiol Learn Mem. 2008; 89 :324–337. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Helmstetter FJ, Tershner SA. Lesions of the periaqueductal gray and rostral ventromedial medulla disrupt antinociceptive but not cardiovascular aversive conditional responses. J Neurosci. 1994; 11 (Pt 2):7099–7108. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Ji G, Neugebauer V. Hemispheric lateralization of pain processing by amygdala neurons. J Neurophys. 2009; 102 :2253–2264. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Johansen JP, Tarpley JW, LeDoux JE, Blair HT. Neural substrates for expectation-modulated fear learning in the amygdala and periaqueductal gray. Nat Neurosci. 2010 in press. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Jordan LM. Initiation of locomotion in mammals. Ann N Y Acad Sci. 1998; 860 :83–93. [ PubMed ] [ Google Scholar ]
Kapp BS, Whalen PJ, Supple WF, Pascoe JP, Aggleton JP. Amygdaloid contributions to conditioned arousal and sensory information processing. In: Aggleton JP, editor. The amygdala: Neurobiological aspects of emotion, memory, and mental dysfunction. Wiley-Liss, Inc.; New York: 1992. pp. 229–254. [ Google Scholar ]
Keay KA, Feil K, Gordon BD, Herbert H, Bandler R. Spinal afferents to functionally distinct periaqueductal gray columns in the rat: an anterograde and retrograde tracing study. J Comp Neurol. 1997; 385 :207–29. [ PubMed ] [ Google Scholar ]
Keay KA, Bandler R. Parallel circuits mediating distinct emotional coping reactions to different types of stress. Neurosci Biobehav Rev. 2001; 25 :669–678. [ PubMed ] [ Google Scholar ]
Kim JJ, Rison RA, Fanselow MS. Effects of amygdala, hippocampus, and periaqueductal gray lesions on short- and long-term contextual fear. Behav Neurosci. 1993; 107 :1093–1098. [ PubMed ] [ Google Scholar ]
Lalumiere RT, McGaugh JL. Memory enhancement induced by post-training intrabasolateral amygdala infusions of beta-adrenergic or muscarinic agonists requires activation of dopamine receptors: involvement of right, but not left, basolateral amygdala. Learn Mem. 2005; 12 :527–532. [ PMC free article ] [ PubMed ] [ Google Scholar ]
LeDoux JE. Emotion circuits in the brain. Annu Rev Neurosci. 2000; 23 :155–184. [ PubMed ] [ Google Scholar ]
LeDoux JE, Farb C, Ruggiero DA. Topographic organization of neurons in the acoustic thalamus that project to the amygdala. J Neurosci. 1990; 10 :1043–1054. [ PMC free article ] [ PubMed ] [ Google Scholar ]
LeDoux JE, Iwata J, Cicchetti P, Reis DJ. Different projections of the central amygdaloid nucleus mediate autonomic and behavioral correlates of conditioned fear. J Neurosci. 1988; 8 :2517–2529. [ PMC free article ] [ PubMed ] [ Google Scholar ]
LeDoux JE, Ruggiero DA, Forest R, Stornetta R, Reis DJ. Topographic organization of convergent projections to the thalamus from the inferior colliculus and spinal cord in the rat. J Comp Neurol. 1987; 264 :123–146. [ PubMed ] [ Google Scholar ]
Lee T, Kim JJ. Differential effects of cerebellar, amygdalar, and hippocampal lesions on classical eyeblink conditioning in rats. J Neurosci. 2004; 24 :3242–3250. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Leman S, Dielenberg RA, Carrive P. Effect of dorsal periaqueductal gray lesion on cardiovascular and behavioral responses to contextual conditioned fear in rats. Behav Brain Res. 2003; 143 :169–176. [ PubMed ] [ Google Scholar ]
Liebman JM, Mayer DJ, Liebeskind JC. Mesencephalic central gray lesions and fear-motivated behavior in rats. Brain Res. 1970; 23 :353–370. [ PubMed ] [ Google Scholar ]
Mantyh PW. Forebrain projections to the periaqueductal gray in the monkey, with observations in the cat and rat. J Comp Neurol. 1982; 206 :146–158. [ PubMed ] [ Google Scholar ]
Maren S. Synaptic mechanisms of associative memory in the amygdala. Neuron. 2005; 47 :783–786. [ PubMed ] [ Google Scholar ]
McDonald AJ. Organization of amygdaloid projections to the prefrontal cortex and associated striatum in the rat. Neuroscience. 1991; 44 :1–14. [ PubMed ] [ Google Scholar ]
Milad MR, Rauch SL, Pitman RK, Quirk GJ. Fear extinction in rats: Implications for human brain imaging and anxiety disorders. Biol Psychol. 2006; 73 :61–71. [ PubMed ] [ Google Scholar ]
Miller MM, McEwen BS. Establishing an agenda for translational research on PTSD. Ann NY Acad Sci. 2006; 1071 :521–524. [ PubMed ] [ Google Scholar ]
Mobbs D, Petrovic P, Marchant JL, Hassabis D, Weiskopf N, Seymour B, Dolan RJ, Frith CD. When fear is near: threat imminence elicits prefrontal-periaqueductal gray shifts in humans. Science. 2007; 317 :1079–1083. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Mobbs D, Marchant JL, Hassabis D, Seymour B, Tan G, Gray M, Petrovic P, Dolan RJ, Frith CD. From threat to fear: he neural organization of defensive fear systems in humans. J Neurosci. 2009; 29 :12236–12243. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Moita MA, Rosis S, Zhou Y, LeDoux JE, Blair HT. Hippocampal place cells acquire location-specific responses to the conditioned stimulus during auditory fear conditioning. Neuron. 2003; 37 :485–497. [ PubMed ] [ Google Scholar ]
Moita MA, Rosis S, Zhou Y, LeDoux JE, Blair HT. Putting fear in its place: remapping of hippocampal place cells during fear conditioning. J Neurosci. 2004; 24 :7015–7023. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Mongeau R, Miller GA, Chiang E, Anderson DJ. Neural correlates of competing fear behaviors evoked by an innately aversive stimulus. J Neurosci. 2003; 23 :3855–3868. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Neugebauer V. Subcortical processing of nociceptive information: Basal ganglia and amygdala. Handb Clin Neurol. 2006; 81 :141–58. [ PubMed ] [ Google Scholar ]
Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. Academic Press: Sydney. 1997 [ Google Scholar ]
Rau V, DeCola JP, Fanselow F. Stress-induced enhancement of fear learning: An animal model of posttraumatic stress disorder. Neurosci Biobehav Rev. 2005; 29 :1207–1223. [ PubMed ] [ Google Scholar ]
Rauch SL, Shin LM, Phelps EA. Neurocircuitry models of posttraumatic stress disorder and extinction: Human neuroimaging research—past, present, and future. Biol Psychiatry. 2006; 60 :376–82. [ PubMed ] [ Google Scholar ]
Rescorla RA. Pavlovian conditioned inhibition. Psychol. Bull. 1969; 72 :77–94. [ Google Scholar ]
Rizvi TA, Ennis M, Behbehani MM, Shipley MT. Connections between the central nucleus of the amygdala and the midbrain periaqueductal gray: topography and reciprocity. J Comp Neurol. 1991; 303 :121–131. [ PubMed ] [ Google Scholar ]
Rogan MT, Leon KS, Perez DL, Kandel ER. Distinct neural signatures for safety and danger in the amygdala and striatum of the mouse. Neuron. 2005; 46 :309–320. [ PubMed ] [ Google Scholar ]
Seymour B, Dolan R. Emotion, decision making, and the amygdala. Neuron. 2008; 58 :662–671. [ PubMed ] [ Google Scholar ]
Tarpley JW, Shlifer IG, Birnbaum MS, Halladay LR, Blair HT. Bilateral phosphorylation of ERK in the lateral and centrolateral amygdala during unilateral storage of fear memories. Neuroscience. 2009; 164 :908–917. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Vianna DM, Landeira-Fernandez J, Brandao ML. Dorsolateral and ventral regions of the periaqueductal gray matter are involved in distinct types of fear. Neurosci Biobehav Rev. 2001; 25 :711–719. [ PubMed ] [ Google Scholar ]
Walker DL, Cassella JV, Lee Y, De Lima TC, Davis M. Opposing roles of the amygdala and periaqueductal gray in fear-potentiated startle. Neurosci Biobehav Rev. 1997; 21 :743–753. [ PubMed ] [ Google Scholar ]
Weiskrantz L. Behavioral changes associated with ablation of the amygdaloid complex in monkeys. J Comp Physiol Psychol. 1956; 49 :381–391. [ PubMed ] [ Google Scholar ]
Zhao Z, Davis M. Fear-potentiated startle in rats is mediated by neurons in the deep layers of the superior colliculus/deep mesencephalic nucleus of the rostral midbrain through the glutamate non-NMDA receptors. J Neurosci. 2004; 24 :10326–10334. [ PMC free article ] [ PubMed ] [ Google Scholar ]

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Evidence for recovery of fear following immediate extinction in rats and humans

Daniela Schiller 1 , 2 , 3 ,
Christopher K. Cain 2 , 3 ,
Nina G. Curley 1 ,
Jennifer S. Schwartz 1 ,
Sarah A. Stern 2 ,
Joseph E. LeDoux 2 , and
Elizabeth A. Phelps 1 , 2 , 4
1 Psychology Department, New York University, New York, New York 10003, USA;
2 Center for Neural Science, New York University, New York, New York 10003, USA

↵ 3 These authors contributed equally to this work.

Fear responses can be eliminated through extinction, a procedure involving the presentation of fear-eliciting stimuli without aversive outcomes. Extinction is believed to be mediated by new inhibitory learning that acts to suppress fear expression without erasing the original memory trace. This hypothesis is supported mainly by behavioral data demonstrating that fear can recover following extinction. However, a recent report by Myers and coworkers suggests that extinction conducted immediately after fear learning may erase or prevent the consolidation of the fear memory trace. Since extinction is a major component of nearly all behavioral therapies for human fear disorders, this finding supports the notion that therapeutic intervention beginning very soon after a traumatic event will be more efficacious. Given the importance of this issue, and the controversy regarding immediate versus delayed therapeutic interventions, we examined two fear recovery phenomena in both rats and humans: spontaneous recovery (SR) and reinstatement. We found evidence for SR and reinstatement in both rats and humans even when extinction was conducted immediately after fear learning. Thus, our data do not support the hypothesis that immediate extinction erases the original memory trace, nor do they suggest that a close temporal proximity of therapeutic intervention to the traumatic event might be advantageous.

Fear conditioning is a widely studied laboratory paradigm for investigating psychological and neural mechanisms of emotional learning in animals, including humans ( LaBar et al. 1998 ; Fendt and Fanselow 1999 ; LeDoux 2000 ; Davis and Whalen 2001 ; Maren and Quirk 2004 ; Phelps et al. 2004 ; LaBar and Phelps 2005 ; Olsson et al. 2005 ). In a typical experiment, a neutral conditional stimulus (CS), such as a tone or image, is paired in time with an aversive unconditional stimulus (US), often an electrical shock. After conditioning, the CS elicits a fear state consisting of behavioral, autonomic, and endocrine responses.

After conditioning, fear of the CS can be reduced or eliminated with an extinction procedure consisting of repeated presentations of the CS without the aversive US. Extinction is believed to induce new inhibitory learning that suppresses fear expression but leaves the original CS–US memory trace intact (for review, see Myers and Davis 2002 ). Evidence for this comes mainly from behavioral studies demonstrating that CS-elicited fear can return after extinction, an impossibility if extinction caused erasure of the original association. The most commonly cited behavioral phenomena supporting this inhibitory learning hypothesis are spontaneous recovery (SR), reinstatement, and renewal. In SR, CS fear re-emerges after extinction with the passage of time ( Pavlov 1927 ; Baum 1988 ; Rescorla 2004 ). In reinstatement, unsignaled exposure to the US after extinction leads to context-dependent return of CS fear ( Rescorla and Heth 1975 ; Bouton and Bolles 1979a ; Westbrook et al. 2002 ). In renewal, CS fear returns when the CS is presented outside of the extinction context ( Bouton and Bolles 1979b ; Bouton and King 1983 ).

Despite this evidence for fear recovery, some reports suggest that extinction may induce partial or complete erasure of the CS–US memory trace. Molecular and physiological studies indicate that extinction may depend on phosphatase activity that reverses neural plasticity thought to mediate fear acquisition learning ( Lin et al. 2003a , b ). More recently, it has been suggested that extinction may induce erasure, inhibitory learning, or both, depending on the maturity of the fear acquisition memory. Myers et al. (2006) , using rats in a fear-potentiated startle (FPS) paradigm, reported that extinction conducted 10 min after fear acquisition produced a loss of CS fear that did not show SR, reinstatement, or renewal. However, extinction conducted days after acquisition led to an initial loss of CS fear that did show recovery. Extinction conducted 1 h post-acquisition produced a loss of fear that only partially recovered. These findings led to the hypothesis that extinction conducted shortly after acquisition erases or prevents consolidation of initial fear learning, whereas extinction of well-consolidated fear learning generates a new inhibitory memory and leaves the original association intact. Extinction conducted between these two extremes, when the fear memory is partially consolidated, may lead to some erasure and some inhibitory learning. Other recent data also suggest that immediate and delayed extinction depend on different mechanisms ( Cain et al. 2005 ).

Fear extinction research inspired the development of behavior therapy for human anxiety ( Wolpe 1969 ; Rauch et al. 2006 ), and nearly all forms of behavioral therapy rely, at least partially, on extinction learning through exposure to fear-arousing stimuli in a safe context ( Craske 1999 ). In addition, anxiety disorders may be characterized by deficiencies in extinction learning ( Jacobs and Nadel 1985 ; Quirk and Gehlert 2003 ). Thus, findings from extinction research are widely believed to have clear and important implications for the treatment of human anxiety such as post-traumatic stress disorder.

One area of considerable controversy relates to the timing of therapeutic intervention following a traumatic experience. Some suggest that very early treatments such as “debriefings,” which involve talking about trauma-associated cues in a safe setting, blunt the long-term impact of psychological trauma ( Everly and and Mitchell 1999 ; Campfield and Hills 2001 ). However, others advocate delaying therapeutic intervention until stress related to the recent trauma has subsided ( Bisson et al. 1997 ; McNally et al. 2003 ; Rothbaum and Davis 2003 ; Gray and Litz 2005 ; Maren and Chang 2006 ). Given that recovery of fear following successful extinction represents a major shortcoming of current therapeutic approaches ( Jacobs and Nadel 1985 ), the possibility that immediate intervention prevents long-term fear recovery may outweigh concerns about exacerbating post-traumatic stress symptoms.

Following the Myers et al. (2006) finding, several laboratories began to explicitly test whether or not immediate extinction produces suppression of CS fear that does not recover. As a first follow up, Maren and Chang (2006) examined the efficacy of immediate extinction, which is necessary to demonstrate any recovery effects. They found that immediate extinction conducted in the context of acquisition is difficult to obtain in rats, which tempers the claim that immediate extinction can produce erasure. Recent human ( Alvarez et al. 2007 ) and unpublished rat (A.M. Woods and M.E. Bouton, unpubl.) studies demonstrated intact renewal of CS fear after immediate extinction. In addition, there are existing studies that conducted extinction the same day as fear acquisition and reported recovery effects as measured by reinstatement, spontaneous recovery, and renewal ( Quirk 2002 ; Phelps et al. 2004 ; LaBar and Phelps 2005 ; Milad et al. 2005 , 2007 ; Kalisch et al. 2006 ; Dirikx et al. 2007 ). However, these latter studies were not designed to directly address the immediate extinction debate, allowing for parametric and procedural differences to account for the recovery effect. In the present studies, we directly examined the hypothesis that immediate extinction leads to fear suppression that does not recover with time (SR) or with unsignaled US exposure (reinstatement). Given the clinical implications of these phenomena, we chose to study both rat and human subjects. In addition, we measured two fear responses that differed from the Myers et al. (2006) study, freezing (in rats) and galvanic skin-conductance responses (in humans), to shed light on the generality of their finding. Contrary to the Myers et al. (2006) study, we found strong evidence for spontaneous recovery and reinstatement in both rats and humans following immediate extinction.

Reinstatement experiment

Previous studies in our laboratory have developed a human reinstatement paradigm and shown that immediately extinguished conditioned fear can be recovered following reinstatement ( LaBar and Phelps 2005 ). This effect was demonstrated using a single cue delay procedure as well as a delay discrimination procedure, both with a full reinforcement schedule, using skin-conductance responding (SCR) as the index of fear. This set of experiments showed that reinstatement in humans is context dependent and cue specific. That is, it does not generalize to other contexts or to nonpredictive cues. Another study also demonstrated the reinstatement effect after immediate extinction in humans using US expectancy and fear ratings ( Hermans et al. 2005 ). In those experiments, however, the reinstatement and fear recovery tests were immediately preceded by extinction and acquisition (i.e., all stages were conducted in the same day). Myers at al. (2006) demonstrated fear erasure following immediate extinction using a different timeline, such that reinstatement was several days apart from acquisition followed by the recovery test 24 h later. This training protocol allows extinction learning to be fully consolidated prior to reinstatement, and in turn, reinstatement learning to be fully consolidated prior to the fear recovery test. Here, we developed a procedure comparable to that of Myers et al. (2006) , in which the stages of extinction, reinstatement, and recovery test were separated by 24 h, thus eliminating a time-dependent confound. We used reinstatement in a different context ( Fig. 1 ) as a control for the reinstatement effect, which has been shown to be contextually mediated in both humans and rats ( Bouton and Bolles 1979a ; Bouton and King 1983 , 1986 ; Bouton 1984 ; Bouton and Peck 1989 ; Frohardt et al. 2000 ; Westbrook et al. 2002 ; LaBar and Phelps 2005 ).

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Illustrations of the experimental contexts in the reinstatement paradigm. ( A ) Context A was a typical laboratory setting in a windowless room with bare white walls containing an office desk and a chair. ( B ) Context B was located in another building and designed as a more domestic room with windows, colorful fabrics, a floor rug, pillows, wooden chairs, posters, several plants and flower arrangements, some scented candles, and classical music in the background. Also, a different experimenter, whom subjects did not meet in day 1 or 3, guided the subjects in this context.

Acquisition and extinction

Figure 2 presents the mean (±SEM) conditioned response as measured by SCR for both same and different context groups in the different experimental stages. During acquisition, responses to the CS were significantly greater than zero in both same ( t (15) = 5.67, P < 0.001) and different context ( t (17) = 7.42; P < 0.001) groups. In each group, this conditioned responding significantly decreased during extinction (paired two-tailed t -tests; same, t (15) = 3.53, P < 0.01; different, t (17) = 4.37, P < 0.001). The responses in the last trial of extinction were not significantly different from zero (same, t (15) = 2.08; different, t (17) = 1.67). There was no significant difference between the same and different context groups in acquisition or extinction (independent two-tailed t -tests; acquisition, t (32) = 1.08; extinction, t (32) = 0.44). These results show that fear was successfully acquired and extinguished on day 1, in both same and different context groups at an equivalent level.

Reinstatement of CS fear following immediate extinction in humans. Mean (±SEM) conditioned responses by context condition and experimental phase are presented. Both the same and different context groups showed acquisition of the conditioned fear that significantly decreased with extinction. The reinstatement phase occurred at the time point indicated by the vertical dashed line. The recovery test (between vertical solid lines), assessing reinstatement, compared the last trial of extinction (before reinstatement) and the first trial of re-extinction (after reinstatement). Although recovery of immediately extinguished fear occurred in both groups regardless of the context of reinstatement, the recovery was more robust when reinstatement took place in the same context as that of acquisition and extinction. This was supported by a more significant recovery in the same versus different context, as well as a significant difference in the first re-extinction response between the same and different context groups. Importantly, this was the only difference found between the two groups (* P < 0.05; ** P < 0.01; *** P < 0.001; ^ denotes comparison between acquisition and extinction within group; † denotes recovery test within group).

Recovery test

The results of the recovery test assessing reinstatement are presented in Figure 2 (between vertical solid lines). Recovery of immediately extinguished fear occurred in both groups regardless of the context of reinstatement. Specifically, there was a significant difference between the conditioned response in the last trial of extinction (before reinstatement) compared with the first trial of re-extinction (after reinstatement), in both same (paired two-tailed t -test; t (15) = 5.42, P < 0.001) and different context (paired two-tailed t -test; t (17) = 2.52; P < 0.05) groups. However, the recovery was more robust when reinstatement took place in the same context as acquisition and extinction. This was supported by the more significant recovery in the same versus different context ( P < 0.001 vs. P < 0.05, respectively), as well as a significant difference in the first re-extinction response between the same and different context groups (independent two-tailed t -test; t (32) = 2.94, P < 0.01). Importantly, this was the only difference found between the two groups. These results show that conditioned fear that is immediately extinguished in humans can be successfully recovered following reinstatement.

Spontaneous recovery (SR) experiment

To study SR in humans we used a partial reinforcement discrimination paradigm. In addition to examining the SR effect, we also sought to test whether this effect was cue specific or whether it would generalize to nonpredictive cues because of a general arousal effect. The use of a discrimination design, which is different from the single CS design used in the reinstatement experiment described above, allowed us to generalize the recovery effect across conditioning procedures, verifying that recovery following immediate extinction is not dependent on the particular conditioning parameters used.

The mean differential SCR (CS+ minus CS−) during the different stages of the procedure is presented in Figure 3 . The differential SCR was significantly greater than zero in acquisition ( t (13) = 2.50, P < 0.05) and early extinction ( t (13) = 2.55, P < 0.05). This differential responding significantly decreased from early to late extinction (paired two-tailed t -test; t (13) = 2.19, P < 0.05) and by late extinction was no longer significantly different from zero ( t (13) = 1.26). These results show that conditioned fear was successfully acquired and extinguished on day 1.

Spontaneous recovery of immediately extinguished fear in humans. Mean (±SE) differential SCR (CS+ minus CS−) in each experimental phase is presented. A difference between responses to the CS+ compared to CS− was present during acquisition and immediate extinction but decreased by delayed extinction. The spontaneous recovery test (between vertical solid lines) compared the differential SCR with the first CS+ and CS− presented in re-extinction with the differential SCR to the last CS+ and CS− presented in extinction. Extinction and re-extinction were separated by 24 h (indicated by the vertical dashed line). As can be seen, the differential responding in the last trial of extinction was not significantly different from zero, whereas the differential responding 24 h later was. This increase in differential responding (recovery test) was statistically significant (* P < 0.05; ‡ denotes comparison of the differential conditioned response to zero; ^ denotes comparison between early and late extinction; † denotes recovery test; n.s. , not significantly different from zero).

The results of the SR test are presented in Figure 3 (between vertical solid lines). This test compared the differential SCR to the first CS+ and CS− presented in re-extinction with the differential SCR and the last CS+ and CS− presented in extinction. The two phases of extinction and re-extinction were separated by 24 h (indicated by the vertical dashed line in Fig. 3 ). This comparison yielded a significant difference (paired two-tailed t -test; t (13) = 2.31; P < 0.05). Moreover, the differential responding in the last trial of extinction was not significantly different from zero ( t (13) = 0.05) whereas the differential responding 24 h later was ( t (13) = 2.19, P < 0.05). These results show that conditioned fear that is immediately extinguished in humans can spontaneously recover with the passage of time. Importantly, this effect was cue specific and did not generalize to nonpredictive cues. Thus, it cannot be attributed to a general arousal effect exhibited by elevated SCR, since such an effect would have not discriminated the predictive from the nonpredictive cue.

Reinstatement of conditioned fear was compared after immediate or delayed extinction with six experimental groups. There were three groups of rats in each condition: NE-R (no extinction–reinstatement), E-NR (extinction–no reinstatement), and E-R (extinction–reinstatement). The success of extinction was assessed at the post-extinction test. The return of conditioned fear after reinstatement shocks was assessed at the post-reinstatement test. Comparisons between no-extinction and extinction groups at the post-extinction test demonstrate long-term extinction memory in each condition. Comparisons between no-reinstatement and reinstatement groups at the post-reinstatement test demonstrate recovery of fear following unsignaled shocks in the test context. Comparisons between the post-extinction test and the post-reinstatement test in the NE-R groups demonstrate summation of CS− and context fear following unsignaled shocks in the test context.

Acquisition of conditioned fear was identical in immediate and delayed extinction groups; freezing did not differ during the third conditioning trial of acquisition with data combined for both experiments ( t (58) = 0.6, P = 0.56; Fig. 4 ). Note also that there were no differences between any of the individual groups in freezing during this third acquisition CS ( P- values > 0.05). The acquisition data for four rats were lost because of an error recording in one session, and these rats were excluded from the acquisition analysis but remained in the study for the rest of the experiment. Freezing prior to the first CS was near zero in both immediate and delayed extinction rats ( t (46) = 1.0, P = 0.30). Within-session extinction differed between immediate and delayed extinction conditions; a two-way ANOVA revealed significant effects of group, trial, and the interaction (group: F (1920) = 91.7, P < 0.001; time: F (19,920) = 19.1, P < 0.001; group × time: F (19,920) = 2.8, P < 0.001). Immediate extinction rats froze slightly less than delayed extinction rats at the outset of extinction training; however, freezing was not significantly different between the groups during the first extinction CS ( P > 0.05). During the course of extinction, delayed extinction rats froze considerably more than immediate extinction rats, especially during CSs 3–9 ( P values < 0.05). However, by the end of extinction training, rats in both groups showed near complete loss of CS-elicited freezing.

Rat fear acquisition and within-session extinction learning for all immediate extinction (open circles) and delayed extinction (closed circles) groups. Rats were presented with 20 nonreinforced CS presentations in a novel context immediately following fear acquisition (immediate extinction) or 3 d following fear acquisition (delayed extinction). Both groups showed equivalent levels of freezing during the third acquisition CS (acq.) and freezing was low prior to the first extinction CS (pre). Immediate extinction rats generally froze less and extinguished faster than delayed extinction rats. For the acquisition time point: n = 30 rats/group. For the extinction session time points: n = 24 rats/group. Note that data were combined from the rat reinstatement experiment and spontaneous recovery experiment, since all animals within each group were treated identically through this phase. The data for four rats (two immediate extinction and two delayed extinction) were lost for the acquisition time point because of a recording error. * P < 0.05 compared with immediate extinction.

Post-extinction and post-reinstatement tests

Post-extinction and post-reinstatement tests were analyzed in separate two-way ANOVAs (group × test) for the immediate and delayed extinction conditions ( Fig. 5 ). The two-way ANOVA in the immediate extinction condition revealed significant effects of group, test, and the interaction (group: F (2,21) = 25.4, P < 0.001; test: F (1,21) = 5.9, P < 0.05; group × test: F (2,21) = 9.1, P < 0.01). Long-term extinction was evident in the post-extinction test; the E-NR and E-R groups froze significantly less than the NE-R group ( P -values < 0.01). Reinstatement of conditioned fear was also evident in the post-reinstatement test; rats in the E-R group froze significantly more during the test CSs than rats in the E-NR group ( P < 0.05) and no differently from rats in the NE-R condition. Importantly, summation of CS fear and context fear (due to the unsignaled reinstating USs) was minimal; freezing in the NE-R group did not significantly differ between the post-extinction and post-reinstatement tests ( t (7) = 0.6, P = 0.56). The two-way ANOVA for the delayed extinction condition revealed an identical pattern of results (group: F (2,21) = 11.7, P < 0.001; test: F (1,21) = 6.0, P < 0.05; group × test: F (2,21) = 6.4, P < 0.01). Long-term extinction of freezing was evident ( P values < 0.05 for extinction groups vs. the no-extinction group), CS freezing reinstated after unsignaled USs ( P < 0.05, E-NR vs. E-R), and summation of CS and context fear was minimal ( t (7) = 1.4, P = 0.21, post-extinction vs. post-reinstatement test for NE-R group).

Evidence for reinstatement of CS fear following immediate or delayed extinction in rats. ( A ) Mean freezing during the post-extinction test ( left , Post-Ext. test) and the post-reinstatement test ( right , Post-Reinst. test) for all rats in the immediate extinction condition. ( B ) Mean freezing during the post-extinction test ( left , Post-Ext. test) and the post-reinstatement test ( right , Post-Reinst. test) for all rats in the delayed extinction condition. Reinstatement of CS fear during the post-reinstatement test was evident regardless of whether extinction occurred 12 min or 3 d following fear acquisition. Note also that summation of CS and context fear does not account for return of CS-elicited fear in the ext.–reinst. group; summation was minimal (compare CS-elicited freezing in the no-ext.–reinst. groups between the post-extinction test and the post-reinstatement test). (Hatched bars) No extinction–reinstatement; (open bars) extinction–no reinstatement; (solid bars) extinction–reinstatement. * P < 0.05 vs. ext.–no-reinst. group, + P < 0.05 vs. no-ext.–reinst. group.

Spontaneous recovery after immediate versus delayed extinction was examined with two groups of rats; immediate extinction rats received 20 nonreinforced CS presentations ∼12 min after the acquisition session ended, whereas delayed extinction rats received the same extinction three days after acquisition.

Acquisition

Freezing was assessed during the third acquisition CS, all 20 extinction CSs, and a single SR test CS. There was no difference between groups in freezing during the third acquisition CS, suggesting that fear conditioning was equivalent in the immediate and delayed extinction conditions ( t (14) = 0.2, P = 0.89).

As noted above, immediate extinction rats froze slightly less than delayed extinction rats during the first extinction CS and showed a more rapid decline in freezing during extinction training. However, both groups showed similarly low freezing by the end of the extinction session.

Twenty-one days later, all rats were returned to Context B for a single SR test CS. SR was assessed by comparing freezing during the last extinction CS with freezing during the spontaneous recovery test CS with a two-way group (immediate vs. delayed extinction) × time (extinction end vs. spontaneous recovery test) ANOVA. There were no differences in freezing between the groups overall (group: F (1,14) = 1.0, P = 0.35) or during either of these two CSs ( P -values > 0.05). Spontaneous recovery of CS-elicited freezing was evident in both groups (time: F (1,14) = 0.9, P < 0.01) and again this recovery did not differ between the groups (group × time: F (1,14) = 0.3, P = 0.63).

We tested the hypothesis that extinction conducted immediately after fear conditioning results in suppression of CS fear that does not recover with time (spontaneous recovery) or with unsignaled US presentations (reinstatement). We examined this hypothesis with both rats and human subjects, using different dependent measures of conditioned fear: freezing in rats and SCR in humans. We found strong evidence for recovery of fear following immediate extinction in all experiments. In the human immediate extinction experiments, allowing a 24-h delay between extinction and testing, or presenting the subjects with unsignaled USs, led to CS fear that was nearly equivalent to pre-extinction levels ( Figs. 2 , 3 ). The rat experiments allowed for a direct comparison between immediate and delayed extinction. We found no evidence that reinstatement or spontaneous recovery was weaker following immediate extinction. In both experiments, rats in the immediate extinction group froze at least as much as rats in the delayed-extinction group during the critical test ( Figs. 5 , 6 ). Thus, our data do not support the general hypothesis that immediate extinction erases or prevents the consolidation of recently acquired conditioned fear.

Evidence for spontaneous recovery following immediate or delayed extinction in rats. Mean freezing for immediate (open bars) and delayed extinction (solid bars) groups during the last extinction session CS ( left ) and the spontaneous recovery test session CS ( right , 21 d later). Both groups showed significant spontaneous recovery of CS fear and the groups did not freeze differently at the end of extinction or during the SR test CS. * P < 0.05 for time in a two-way group × time (Ext. End vs. SR Test) ANOVA.

The rat experiments also allowed us to examine within-session extinction learning rates with a considerable degree of statistical power, which is typically not possible in the FPS paradigm because the fear response is measured only after and not during the extinction session. Since rats in the immediate and delayed extinction conditions were treated identically through the end of the extinction training we combined the groups for analysis ( Fig. 4 ). During extinction training, there was a nonsignificant trend toward higher freezing during the first extinction CS in the delayed extinction group and a large difference in freezing between the groups as training progressed. Immediate extinction rats froze less throughout the session and appeared to extinguish faster; however, it is likely that this reflects differences in performance rather than learning. Rats in the immediate and delayed groups froze the same during the third CS–US pairing of acquisition, suggesting that learning was equivalent in the two groups. And, as noted above, spontaneous recovery and reinstatement were equivalent in the immediate and delayed groups, a result that might not be expected if the immediate extinction rats had weaker initial learning or disrupted consolidation. Such a performance effect is not entirely surprising. For instance, Wagner’s SOP and AESOP models ( Wagner 1978 , 1981 ; Wagner and Brandon 1989 ) predict that freezing will be disrupted at the outset of immediate, but not delayed, extinction even though rats in these groups may have equivalent CS–US associations. However, these models also predict that immediate extinction would be impaired relative to delayed extinction, which is not supported by our data. Clearly, this large behavioral effect requires further study. However, we are confident that animals in both conditions had strong CS–US associations from acquisition and strong fear extinction (see last extinction trial [ Fig. 4 ] and the post-extinction tests [ Fig. 5 ]).

The present experiments were designed to assess whether the finding of Myers et al. (2006) would apply to a different behavioral measure in rats and would extend to humans. However, we were unable to demonstrate a difference in fear recovery between the immediate and delayed extinction conditions in rats. In humans, a number of studies have demonstrated fear recovery following immediate extinction, although these studies were not designed to directly address this question ( Phelps et al. 2004 ; LaBar and Phelps 2005 ; Milad et al. 2005 , 2007 ; Kalisch et al. 2006 ; Dirikx et al. 2007 ). Our findings in humans confirm and extend these existing data, directly demonstrating the return of the fear response due to spontaneous recovery and reinstatement following immediate extinction.

There are a number of differences between the study of Myers et al. (2006) and our own that could potentially account for the discrepancy. In the rat studies, we examined freezing rather than FPS as a measure of conditioned fear and used tone instead of light CSs. The procedure, stimuli, subjects, and dependent measure were all vastly different for the human studies, and any one of these differences could also account for the found discrepancy with the results of Myers et al. (2006) . However, our findings are internally consistent (similar results in rats and humans) and are supported by several other lines of evidence. Studies previously demonstrated spontaneous recovery when extinction is conducted 1 h after fear acquisition ( Quirk et al. 2000 ; Quirk 2002 ), consistent with our immediate extinction findings and partially at odds with those of Myers et al. (2006) . An unpublished report showed intact renewal of CS fear when extinction was conducted 15 min after acquisition (A.M. Woods and M.E. Bouton, unpubl.). Alvarez et al. (2007) report intact renewal of CS fear after immediate extinction in humans, and this effect was demonstrated with two dependent measures (SCR and FPS). Reinstatement of CS fear after immediate extinction has also been indirectly implied by human studies that were not designed to examine this issue ( Hermans et al. 2005 ; LaBar and Phelps 2005 ). Thus, the lack of fear recovery following immediate extinction may be a nuanced effect rather than a general property of extinction.

At first glance, our data may seem at odds with another study investigating immediate extinction. Maren and Chang (2006) report that recently acquired fear is resistant to extinction conducted 15 min later compared with extinction conducted 24 h later (using a conditioned fear paradigm in rats and measuring freezing). Importantly, they show that rats fail to learn extinction if CS-alone presentations begin immediately after acquisition and occur in the acquisition context. They did not explicitly examine fear recovery effects after extinction with this protocol because immediately extinguished rats do not learn extinction in the first place. They do show effective immediate extinction in a different context but do not report CS-fear recovery data. In our rat experiments, extinction took place in a different context than acquisition, and in this condition, immediate extinction is at least as effective as delayed extinction. There was no delayed extinction condition in our human experiments so we cannot comment on whether or not delayed extinction is more effective than immediate extinction when all phases are conducted in the same context. Nevertheless, immediate extinction, which was conducted in the acquisition context, was clearly effective. Our ability to manipulate fear in humans is limited for ethical reasons. It is likely that in the human experiments the levels of fear were much lower than those in the rat experiments reported by Maren and Chang (2006) , and thus there was little contextual fear to impede extinction in the humans. Therefore, our data are not at odds with those reported by Maren and Chang (2006) .

In addition to the basic finding that CS fear recovers after immediate extinction, our experiments provide support for two other hypotheses related to reinstatement of extinguished fear. Reinstatement is hypothesized to be context dependent ( Bouton and Bolles 1979a ; Bouton and Peck 1989 ) and to result from changes in the occasion-setting properties of the context, as opposed to summation of CS- and context-elicited fear ( Bouton and King 1983 ). Our human reinstatement experiment examined reinstatement of CS fear in Context A after acquisition and extinction in Context A. We had two different reinstatement groups: one that received unsignaled USs in Context A and one that received unsignaled USs in Context B. As predicted, reinstatement was context specific: Although subjects receiving the USs in either the same or different context as the final test showed a return of CS-elicited fear, this effect was significantly greater in the same context compared with the different context group. Importantly, that was the only difference between these two groups throughout the task ( Fig. 2 ). The design of our rat reinstatement experiment allowed us to comment on the likelihood that summation of context- and CS-elicited fear accounts for reinstatement. Our no-extinction–reinstatement control groups received the same unsignaled US presentations in the extinction/test context but were never extinguished. Comparing freezing between the post-extinction and post-reinstatement tests for these groups ( Fig. 5 ) reveals that summation was minimal and cannot account for the robust reinstatement effect observed for the extinction–reinstatement group. Thus, these findings support the notion that reinstatement is context dependent and results from a process other than summation of CS and context fear.

It should be noted that in our human experiments we cannot be sure that immediate extinction had no detrimental effects on conditioning consolidation. Although we found in both reinstatement and spontaneous recovery experiments that the extinguished fear responses recovered to the same levels of conditioning ( Figs. 2 , 3 ), it is possible that a no-extinction group would have exhibited higher fear levels during fear recall because of an incubation effect ( Eysenck 1968 ). However, a previous renewal study in humans ( Milad et al. 2005 ) found that fear was recovered in an immediate extinction group to similar levels exhibited by a no-extinction group during the fear recall phase. These results indicate that immediate extinction did not interfere with conditioning consolidation, and the fear memory remained intact. Moreover, these results provide no evidence for an incubation effect in humans. In any event, the robust recovery rates we observed in our human experiments strongly argue against the erasure of fear suggested by Myers et al. (2006).

The results of spontaneous recovery in humans allowed us to rule out the possibility that the recovery occurred because of a general arousal effect. Such general elevation in SCR baseline would affect both CS+ and CS− responses indiscriminately. In contrast, we could observe significantly greater responding to the CS+ versus the CS− levels upon return to the experimental context, 24 h after extinction. These results confirm the cue specificity of the spontaneous recovery effect, which is restricted to the CS alone.

Findings from fear extinction research have important implications for the treatment of pathological fear in humans for two related reasons. First, a critical component of nearly all cognitive-behavior therapies for fear disorders is fear extinction ( Wolpe 1969 ; Craske 1999 ; Rauch et al. 2006 ). Thus, findings from extinction studies are likely to directly translate to better treatments for anxious patients. Second, fear disorders may be characterized by impairments in the ability to extinguish learned fear ( Quirk and Gehlert 2003 ). Given this, findings from extinction studies may aid in targeting specific neural and psychological processes that are dysfunctional in pathological fear.

An area of considerable controversy regarding cognitive-behavioral therapy following severe trauma is the relative efficacy of immediate versus delayed interventions. Some believe that very early interventions, such as debriefings in a safe setting, aid in the long-term treatment of anxiety perhaps by blunting the traumatic memory or by preventing fear recovery following successful treatment ( Everly and and Mitchell 1999 ; Campfield and Hills 2001 ). However, the available data is mixed regarding the hypothesized benefit of early intervention. Indeed, some have argued that very early interventions exacerbate long-term fear by adding to the stress of the traumatic experience ( Bisson et al. 1997 ; McNally et al. 2003 ; Rothbaum and Davis 2003 ; Gray and Litz 2005 ). If solid evidence existed that immediate extinction produced nonrecovering fear suppression, one could argue that early therapeutic interventions were warranted even if that meant adding to the patient’s immediate level of discomfort. The jury is still out on this important debate, but our data indicate that immediate extinction does not prevent recovery of CS fear in rats and humans and suggest that cognitive-behavior treatments immediately following severe trauma may not be especially advantageous. Immediate and delayed extinction may, however, operate through different neural or molecular mechanisms ( Cain et al. 2005 ), and future studies will be needed to examine the relative efficacy of pharmacological agents on these two processes.

Materials and Methods

Reinstatement Experiment

Forty participants (18 to 27 yr of age) were recruited through posted advertisements. Subjects were excluded from the experiment if on day 1 they showed no measurable SCR response (below minimal response criteria), failure to acquire the conditioned response (mean response of last four acquisition trials was not significantly different from zero), or failure to extinguish the fear response (mean response of last four extinction trials was not significantly different from mean late acquisition response). We employed these criteria because we could not assess fear recovery without a reliable SCR measure and without showing that the conditioned response was successfully acquired and extinguished. These exclusion criteria are widely accepted in the conditioning and extinction literature ( Phelps et al. 2004 ; Milad et al. 2005 ; Olsson et al. 2005 ; Kalisch et al. 2006 ). The final analysis included 34 participants (18 females). The experiment was approved by the University Committee on Activities Involving Human Subjects. All subjects gave informed consent and were paid for their participation.

Reinstatement procedure

A 100% reinforcement paradigm was used. The CS was a fractal image, and the US was a mild shock to the wrist. Two different fractal images were used and counterbalanced across subjects. All CSs in acquisition and extinction were presented for 6 sec with a variable 10- to 12-sec inter-trial interval (ITI). Subjects were instructed to pay attention to the screen and notice whether there is a relationship between the presentation of the images and the shock. There were four stages to the study: acquisition, extinction, reinstatement, and re-extinction. The stages were completed over 3 d as follows: Day 1—Acquisition and extinction . During acquisition, there were four habituation trials and then eight presentations of the CS that co-terminated with the US. Extinction immediately followed acquisition (after the 10- to 12-sec ITI) with 16 nonreinforced presentations of the CS. Day 2—Reinstatement . Twenty-four hours later, subjects received four presentations of the US, with a 50-sec ITI. Subjects were randomly allocated into one of two groups. One group ( n = 16) received the US presentations in the same room as day 1 (context A; Fig. 1A ), while viewing the solid background color of the CS but not the CS itself. The other group ( n = 18) received the presentations of the US in a different room (context B; Fig. 1B ), located in another building and guided by a different experimenter, while viewing a different patterned background. Day 3—Re-extinction . Twenty-four hours later, all subjects returned to the same room that was used on day 1 (context A) and again underwent extinction, consisting of 20 nonreinforced presentations of the CS.

Psychophysiological stimulation and assessment

Mild shocks were delivered through a stimulating bar electrode attached with a Velcro strap to the right inner wrist. A Grass Medical Instruments stimulator charged by a stabilized current was used. Subjects determined the level of the shock themselves, beginning at a very mild level of shock (10 V) and gradually increasing the level until the shock reached the maximum level that they determined was “uncomfortable, but not painful” (the maximum level was 50 V). All shocks were given for 200 msec, with a current of 50 pulses per second. Skin conductance was assessed using two Ag–AgCl electrodes, which were connected to a BioPac Systems skin-conductance module. The electrodes were attached to the first and second fingers of the left hand, between the first and second phalanges.

SCR waveforms were analyzed offline, using AcqKnowledge 3.9 software (BIOPAC Systems Inc.). SCR amplitudes to the CS and US were the dependent measures of conditioned and unconditioned responses, respectively. The level of SCR response was determined by taking the base to peak difference for the first largest waveform (in microsiemens, μs) in the 0.5- to 4.5-sec window following stimulus onset. The minimal response criterion was 0.02 μs. The raw SCR scores were square root transformed to normalize distributions. These normalized scores were scaled according to each subject’s unconditioned response by dividing each response by the mean square-root-transformed US response. This additional step allowed us to account for individual differences in SCR and to compute a relative measure of conditioned response linked to each participant’s unconditioned response ( Olsson et al. 2005 ).

Statistical analysis

Trials were averaged into blocks representing each experimental phase (i.e., acquisition, extinction, and re-extinction). To test for the recovery of fear following reinstatement, data from the first re-extinction trial was compared with that of the last extinction trial ( Rescorla and Heth 1975 ). An alpha level of 0.05 was set for all statistical comparisons.

Spontaneous recovery experiment

Seventeen participants (18 to 28 yr of age) were recruited through posted advertisements. Subjects were excluded from the experiment if on day 1 they showed no measurable SCR response, failure to acquire the conditioned response (no significant mean differential responding to CS+ compared with CS− in last four trials of acquisition), or failure to extinguish the fear response (a significant mean differential responding to CS+ compared with CS− in last four trials of extinction). The final analysis included 14 participants (seven females). The experiment was approved by the University Committee on Activities Involving Human Subjects. All subjects gave informed consent and were paid for their participation.

Spontaneous recovery paradigm

A simple discrimination, partial reinforcement paradigm was used. The CSs were two different colored snake images (red and four yellow), and the US was a mild shock to the wrist. One of the snake images was designated as the CS+, and paired with the shock on 33% of the trials, while the other was never paired with the shock (CS−). We used negatively valenced CSs to enhance overall emotional reactivity ( Morris et al. 1998 ; Critchley et al. 2002 ; Ohman 2005 ; Kalisch et al. 2006 ). Each stimulus served as both CS+ and CS− counterbalanced across subjects. The CSs for acquisition and extinction were presented for 4 sec each with a 12-sec ITI. Subjects were instructed to pay attention to the screen and notice whether there is a relationship between the presentation of the images and the shock. There were three stages to this paradigm: acquisition, extinction, and re-extinction. The stages were completed over 2 d as follows: Day 1—Acquisition and extinction . Acquisition consisted of nonreinforced presentations of the CS+ and CS− (12 each) intertwined with an additional six presentations of the CS+ that co-terminated with the US. Extinction immediately followed acquisition after the 12-sec ITI, with nonreinforced presentations of the CS+ and CS− (eight each). Day 2—Re-extinction . Twenty-four hours later, subjects returned to the same room that was used on day 1 and again underwent extinction, consisting of nonreinforced presentations of the CS+ and CS− (12 each).

These were the same as reinstatement paradigm (see above).

SCR following the US was analyzed to assess unconditioned responding, but only trials that did not coterminate with the US were analyzed to measure fear acquisition. The conditioned fear response was assessed as the differential SCR, that is, the SCR to the CS+ minus the SCR to the CS−. Trials were averaged into blocks representing the experimental phases. The use of a partial reinforcement procedure allowed for a slow extinction, since extinction occurs rapidly in humans with 100% reinforcement ( LaBar et al. 1998 ; Phelps et al. 2004 ). Thus, the trials of the extinction phase were divided into first half and second half (early and late extinction) to identify a gradual change in responsivity as extinction progresses. To test for the recovery of fear due to passage of time (spontaneous recovery), the differential SCR to the first CS+ and CS− presented in re-extinction was compared with the differential SCR to the last CS+ and CS− presented in extinction. An alpha level of 0.05 was set for all statistical comparisons.

All experiments were conducted on naïve 300–350 g male Sprague-Dawley rats (Hilltop Lab Animals, Inc.) and were approved by NYU’s Animal Care and Use Committee. Rats were maintained on a 12:12 light/dark schedule and allowed free access to food and water. All testing was conducted during the light phase in illuminated testing rooms.

Two contexts (A and B) were used for all behavioral testing. Context A consisted of four standard fear-conditioning chambers (Model E10-10, Coulbourn Instruments). Chambers were constructed of aluminum and Plexiglas walls with stainless steel grid flooring that was attached to a shock generator (Model H13-15; Coulbourn Instruments). Context B consisted of four separate conditioning chambers (ENV-001; MedAssociates, Inc.) located in a different room. Context B chambers also had stainless steel grid flooring attached to scrambled shock generators (Models ENV410B and ENV412). Chambers were enclosed within sound attenuating cubicles. Chambers differed in shape (L × W × H; A: 28.5 cm × 26 cm × 28.5 cm; B: 24.5 cm × 30 cm × 21 cm), lighting (A: dim house light; B: house light plus two bright cue lights always on), odor (A: no odor; B: peppermint soap in floor pan), and flooring (A: 5-mm diameter rods spaced 1.5 cm apart; B: 4-mm diameter rods spaced 1.6 cm apart). Individual video cameras were mounted in the ceiling of each chamber and connected via a quad processor to a standard VCR and monitor for videotaping and scoring of freezing. Delivery of stimuli was controlled with Graphic State 2 Software (Coulbourn Instruments) in Context A and MedPC Software in Context B.

The reinstatement experiment consisted of five phases: acquisition, extinction, post-extinction test, reinstatement, and post-reinstatement test. Acquisition took place in Context A, and all other phases took place in Context B. Acquisition consisted of three CS–US pairings (acclimation period = 5 min; ITI = 5 min; post-conditioning period = 5 min). The CS was a pure tone (30 sec, 80 dB, 5 kHz) and the US was a scrambled footshock (0.7 mA, 1 sec) that coterminated with the CS. Extinction consisted of 20 massed presentations of the CS alone (acclimation period = 2 min; ITI = 5 sec; post-extinction period = 2 min). Reinstatement consisted of three unsignaled US presentations (acclimation period = 5 min; ITI = 2 min; post-shock period = 5 min). The post-extinction and post-reinstatement tests were identical and consisted of five CS alone presentations (acclimation period = 2 min; ITI = 3 min; post-test period = 2 min). Two extinction conditions were examined: immediate extinction and delayed extinction. Within each condition, three separate groups of rats were run ( n = 8 rats/group): (1) no-extinction–reinstatement (NE-R); (2) extinction–no-reinstatement (E-NR); and (3) extinction–reinstatement (E-R). Thus, six groups of rats were run beginning on the same day. All rats received the same acquisition session on day 1. Immediate extinction rats were removed from the acquisition chambers in Context A and immediately moved to the Context B chambers. The two extinction groups then began receiving CS presentations in Context B ∼12–15 min after the final acquisition trial. The no-extinction group just remained in the Context B chambers for an equivalent amount of time. After the extinction session all rats were returned to their home cages in the colony room. All delayed extinction rats were returned to their home cages in the colony room after acquisition and remained there for 3 d. On day 4, the delayed extinction groups were placed in Context B for their extinction session, which was the same as the immediate extinction groups (20 CS presentations for extinction groups, no CS presentations for the no-extinction group). On day 5, all rats were returned to Context B to probe CS-elicited fear during the post-extinction test. On day 6, all rats were returned to Context B for the reinstatement session. Rats in the reinstatement groups received three unsignaled foot shocks; rats in the no-reinstatement groups received no foot shocks but remained in the chambers for an identical period. On day 7, all rats were again returned to Context B to probe CS-elicited fear during the post-reinstatement test. Data for the post-extinction and post-reinstatement tests represent mean freezing for the first three test CS presentations.

Data analysis

Defensive freezing, defined as the absence of all nonrespiratory movement ( Blanchard and Blanchard 1971 ; Fanselow 1980 ), served as the index of fear in all rat experiments. Freezing was manually scored from videotapes/DVDs following behavioral testing, and time spent freezing for each 30-sec CS was converted to a freezing percentage. Behavioral scorers were blind to group specification. Data in graphs represent group means ± SEM. Since all immediate and delayed extinction groups in both experiments were treated identically up to the end of the extinction sessions, data for all animals within each condition were combined for statistical analysis of acquisition learning and within-session extinction learning ( Fig. 4 ). Statistical analysis was conducted with GraphPad Prism (version 4.0). Experimental phases with only two groups and a single measure were analyzed with unpaired two-tailed t -tests. Time course data were analyzed using two-way (group × time) ANOVAs with planned post-hoc Bonferroni tests to compare group differences at individual time points. Differences were considered significant when P < 0.05.

Subjects, apparatus, and data analysis

These were the same as the reinstatement experiment with one exception. Grid floors in Context B were covered with black Plexiglas inserts for all experimental phases conducted in B.

The spontaneous recovery experiment consisted of three phases: acquisition, extinction, and spontaneous recovery test. Acquisition was conducted in Context A and the extinction and spontaneous recovery test phases were conducted in Context B. Immediate and delayed extinction conditions were compared (8 rats/group), as previously. Acquisition and extinction sessions were identical to those reported for the reinstatement experiment. Following three CSUS pairings in A, rats received 20 nonreinforced CS presentation in Context B either 12 min or 3 d following acquisition. The spontaneous recovery test was conducted 21 d after the extinction session for both groups and was identical to the tests of the reinstatement experiment (five nonreinforced CS presentations in B).

Acknowledgments

This work was supported by a Fulbright award to D.S.; NRSA F32 MH077458 grant to C.K.C.; NIH grants R01 MH46516, R37 MH38774, K05 MH067048, P50 MH58911 to J.E.L.; and a James S. McDonnell Foundation and NIH R21 MH072279 grants to E.A.P. The authors thank Rita Jou for technical support and assistance in running the experiments and Leib Litman for assistance with statistical analysis.

↵ 4 Corresponding author.

↵ 4 E-mail liz.phelps{at}nyu.edu ; fax (212) 995-4349.

Article is online at http://www.learnmem.org/cgi/doi/10.1101/lm.909208 .

Received January 15, 2008.
Accepted March 31, 2008.
Alvarez, R.P. ,
Johnson, L. ,
Grillon, C.
Bisson, J.I. ,
Jenkins, P.L. ,
Alexander, J. ,
Bannister, C.
Blanchard, R.J. ,
Blanchard, D.C.
Bouton, M.E.
Bouton, M.E. ,
Bolles, R.C.
Cain, C.K. ,
Godsil, B.P. ,
Campfield, K.M. ,
Hills, A.M.
Craske, M.G.
Critchley, H.D. ,
Mathias, C.J. ,
Dolan, R.J.
Davis, M. ,
Whalen, P.J.
Dirikx, T. ,
Hermans, D. ,
Vansteenwegen, D. ,
Baeyens, F. ,
Everly, G.S. ,
Mitchell, J.T.
Eysenck, H.J.
Fanselow, M.S.
Fendt, M. ,
Frohardt, R.J. ,
Guarraci, F.A. ,
Gray, M.J. ,
Van den Bergh, O. ,
Jacobs, W.J. ,
Kalisch, R. ,
Korenfeld, E. ,
Stephan, K.E. ,
Weiskopf, N. ,
Seymour, B. ,
LaBar, K.S. ,
Phelps, E.A.
Gatenby, J.C. ,
Gore, J.C. ,
LeDoux, J.E. ,
LeDoux, J.E.
Lin, C.-H. ,
Yeh, S.-H. ,
Lu, H.-Y. ,
Gean, P.-W.
Leu, T.-H. ,
Chang, W.-C. ,
Wang, S.-T. ,
Maren, S. ,
Chang, C.-H.
Quirk, G.J.
McNally, R.J. ,
Bryant, R.A. ,
Milad, M.R. ,
Quinn, B.T. ,
Pitman, R.K. ,
Orr, S.P. ,
Fischl, B. ,
Rauch, S.L.
Wright, C.I. ,
Quirk, G.J. ,
Morris, J.S. ,
Ohman, A. ,
Myers, K.M. ,
Ressler, K.J. ,
Olsson, A. ,
Ebert, J.P. ,
Banaji, M.R. ,
Pavlov, I.P.
Phelps, E.A. ,
Delgado, M.R. ,
Nearing, K.I. ,
Gehlert, D.R.
Russo, G.K. ,
Barron, J.L. ,
Rauch, S.L. ,
Shin, L.M. ,
Rescorla, R.A.
Rescorla, R.A. ,
Rothbaum, B.O. ,
Wagner, A.R.
Hulse, S.H. ,
Fowler, H. ,
Honig, W.K.
Spear, N.E. ,
Miller, R.R.
Wagner, A.R. ,
Brandon, S.E.
Klein, S.B. ,
Mowrer, R.R.
Westbrook, R.F. ,
Iordanova, M. ,
McNally, G. ,
Richardson, R. ,
Harris, J.A.

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Published: 25 January 2024

Differential recruitment of brain circuits during fear extinction in non-stressed compared to stress resilient animals

Jiah Pearson-Leary 1 ,
Alexander P. Abramenko 2 ,
Valerie Estela-Pro 1 ,
Elizabeth Feindt-Scott 1 ,
Jason Yan 1 ,
Abigail Vigderman 1 ,
Sandra Luz 1 ,
Debra Bangasser 4 ,
Richard Ross 5 ,
Leszek Kubin 6 &
Seema Bhatnagar 1 , 3

Scientific Reports volume 14 , Article number: 2125 ( 2024 ) Cite this article

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Neuroscience
Stress and resilience

Dysfunctional fear responses in post-traumatic stress disorder (PTSD) may be partly explained by an inability to effectively extinguish fear responses elicited by trauma-related cues. However, only a subset of individuals exposed to traumatic stress develop PTSD. Therefore, studying fear extinction deficits in animal models of individual differences could help identify neural substrates underlying vulnerability or resilience to the effects of stress. We used a rat model of social defeat in which rats segregate into passively and actively coping rats. In previous work, we showed that passively coping rats exhibit disruptions in social interaction whereas actively coping rats do not display behaviors differently from controls, indicating their resilience. Here, adult male rats exposed to 7 days of social defeat were tested for fear extinction, retention of extinction, and persistence of retention using contextual fear and ethologically-relevant fear tests. Passively coping rats exhibited elevated freezing in response to the previously extinguished context. Analyses of cFos expressing cells across select brain regions showed high correlations within dorsal hippocampal subregions, while passively coping rats had high correlations between the dorsal hippocampus CA1 and the central and basolateral subregions of the amygdala. Importantly, although control and actively coping rats showed similar levels of behavioral extinction, there was little similarity between activated structures, suggesting stress resilience in response to chronic social defeat involves an adaptive differential recruitment of brain circuits to successfully extinguish fear memories.

Stressed rats fail to exhibit avoidance reactions to innately aversive social calls

Refinement of the stress-enhanced fear learning model of post-traumatic stress disorder: a behavioral and molecular analysis

Environmental enrichment prevents the late effect of acute stress-induced fear extinction deficit: the role of hippocampal AMPA-GluA1 phosphorylation

Introduction.

Post-traumatic stress disorder (PTSD) is a debilitating mental health condition that develops in a subset of people following exposure to traumatic stress 1 , 2 . PTSD is diagnosed based on clusters of symptoms, including re-experiencing and/or having intrusive memories of trauma, avoidance, negative mood and thoughts, and disruptive levels of hyperarousal 1 , 2 . Not all individuals exposed to traumatic events develop PTSD, however 1 . Active coping behaviors have been shown to reduce the risk of developing PTSD, while more passive behaviors such as social avoidance can increase the risk of developing PTSD 3 . Using repeated social defeat by an aggressive resident rat, we have shown that resilient and vulnerable subpopulations emerge based on the coping strategies employed during defeat stress 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 . Rats passively coping during exposure to an aggressive conspecific exhibit changes in behaviors including reductions in social interaction and increases in immobility during the forced swim test as compared to control animals, indicating their vulnerability to the effects of stress. Actively coping rats display behaviors not different from those of non-stressed controls, indicating their resilience to the effects of stress 5 , 6 , 7 , 8 , 9 , 10 . Thus, studying individual differences in response to repeated social defeat stress could ultimately provide insight into the neural substrates that underlie PTSD and related anxiety disorders.

The dysfunctional fear response that characterizes PTSD may be partly explained by an inability to retain the learned extinction of fear responses to previously traumatic cues 12 , 13 . This disruption in fear extinction retention can occur both for the traumatic event and for fear memories both related and unrelated to the trauma that induced PTSD 14 , 15 , 16 , 17 . Thus, preclinical models in which individual differences in extinction retention are observed would be valuable in identifying how traumatic stress re-organizes brain circuits in subsets of individuals to create extinction-resistant fear memories.

In the current study, we examined fear extinction in rats that exhibited passive or active coping strategies during repeated social defeat. We tested these rats in two different contexts: one in which rats were previously socially-defeated, referred to as ethological fear testing due to its proximity to naturalistic aggression, and another using a standard contextual fear paradigm in which control or socially-defeated rats were exposed to unsignaled foot shocks that produce context-specific conditioned fear 18 . Next, we examined functional connectivity via correlations of numbers of c-Fos-expressing cells between brain regions known to be important in stress, fear, and memory following extinction retention testing to identify putative circuits that may underlie vulnerability vs. resilience to the effects of stress on fear learning. Our findings demonstrate that stress vulnerable/passively coping rats show impairments in the retention of fear extinction. Active coping/resilient rats show successful retention of fear extinction similar to controls, however, the response in active coping rats is likely through an adaptive switch in brain circuits recruited during extinction training.

Experiment 1: ethological fear testing

Rats were socially defeated for 7 days (Fig. 1 A). During each episode of social stress, a rat was placed into the home cage territory of an unfamiliar Long-Evans resident previously screened for high aggression. A typical agonistic encounter resulted in intruder subordination or defeat, signaled by the intruder assuming a supine position for 3 s. After defeat, a wire mesh partition was placed in the cage to prevent physical contact between the resident and intruder but allowing visual, auditory, and olfactory contact for the remainder of the 30 min defeat session. On each day, intruder rats were placed in the home cage of a different resident aggressor for 15 min or until the intruder displayed a supine and frozen defeat posture, whichever occurred first. This time to show the defeat posture was recorded as latency to be defeated. One set of resident rats was used for each cohort of intruder rats. Thus, all intruder rats in a given cohort were exposed to the same set of residents but on a different day. This minimized the impact of variations in aggression across the cohort of resident rats. Daily latencies to be defeated displayed by intruder rats were averaged across the 7 days. If an intruder resisted defeat for 15 min, the resident and intruder were separated with the wire partition for the remainder of the session. Controls were placed behind a wire partition in a novel cage for 30 min daily. Rats were returned to their home cage after each session. To identify passively or actively coping rats, the average latency of each rat over the course of 7 days of defeat was entered into an R script used to perform bootstrap cluster analysis (code available at www.github.com/cookpa/socialdefeat ). The analysis provides probabilities for resilience, with 1 indicating resilience and 0 indicating vulnerability, with 0.5 being the point of delineation between actively and passively coping rats. Latencies clustered at 161 s ± 26.3 s (N = 7) for passive coping rats and 359 s + / − 37.1 s (N = 6) for active coping rats ( t = 4.44, p = 0.001; Fig. 1 C). The latencies for each group are similar to our previous results 5 , 6 , 7 , 8 . To measure fear expression and extinction in an ethologically relevant way, rats were then re-exposed to the cage of the resident by which they were last defeated without the resident in the cage as extinction training (day 8), extinction retention testing (day 9), and persistence of extinction retention (day 20) (Fig. 1 A, B).

Fear extinction in response to environment where rats were previously socially-defeated. ( A ) Experimental design for the testing paradigm. ( B ) Extinction testing protocol to assess freezing when the rat was placed in an empty-resident cage where social defeat had previously occurred 24 h, 48 h or 11 days earlier. ( C ) Latencies to social defeat showing splits expressed by animals tested in this experiment. ( D – F ). Bar graphs showing percent total freezing over the course of the 8 min testing trial over the 3 testing days and results of Tukey’s post hoc tests on significant one-way ANOVAs. ( G – I ), Repeated measures analyses of freezing responses separated into 1 min bins, with time as the repeated measure. At days 8 and 9, there were interaction effects, with post hoc tests revealing differences between passive coping, active coping, and/or control rats at several time points. I. On day 20, there was a main effect of group, as shown by an asterisk indicating that passive coping rats had high freezing relative to active coping and control rats. Post hoc significance symbols: Passive vs control = a, passive vs active = b, active vs non-stressed control = c. * p < 0.05 for main effect shown in I. Control n = 8, passive n = 7, active n = 6.

Experiment 1.1. Both passive coping and active coping rats display higher levels of ethological fear compared to novel cage control rats

Both passively and actively coping stress-exposed rats displayed a higher percentage of time spent freezing during extinction (F 2,17 = 4.8, p = 0.02, Fig. 1 D) as compared to rats that were not exposed to social defeat stress and were instead placed in a novel cage daily for the duration of the social defeat paradigm (novel cage controls). In addition, a repeated measures ANOVA revealed a main effect of time (F 7,119 = 8.85, p = 0.0001) and stress group (F 2,17 = 4.86, p = 0.02) and a significant interaction effect (F 14,119 = 2.43, p = 0.005; Fig. 1 G). The post-hoc tests indicated that passive coping and active coping rats both exhibited increased freezing relative to non-stressed control rats at multiple timepoints (Fig. 1 D, G) during the extinction training phase.

Experiment 1.2. Passive coping rats show reduced retention of extinction and this reduction persists over time

Following extinction training (day 8), we tested freezing in response to the aggressor’s cage on day 9 (extinction retention) and day 20 (persistence of extinction retention) without the aggressor resident rat present. On day 9 during extinction retention testing (Fig. 1 E, H), a two-way repeated measures ANOVA revealed main effects of time (F 7,119 = 8.87, p = 0.0001) and group (F 2,17 = 12.51, p = 0.005), and a significant interaction effect (F 14,119 = 2.14, p = 0.001) were observed). Post-hoc analysis of the main group effect revealed that passive coping rats showed increased freezing compared to non-stressed control rats (Fig. 1 E). Further, post-hoc analyses for differences at individual time points confirmed that passive coping rats had increased freezing relative to active coping rats at time points 4, 5, and 6 (denoted by letters) and increased freezing relative to non-stressed control rats at time points 1–6 (Fig. 1 H). Active coping rats had increased freezing relative to control rats only at time point 2. When examining freezing behaviors during testing for persistence of extinction retention (day 21; Fig. 1 F, I), a two-way repeated measures ANOVA revealed a main effect of group (F 2,17 = 7.715, p = 0.006). Post-hoc analyses revealed that passive coping rats had increased freezing relative to both active coping and control rats. These data suggest that extinction retention deficits persist over time in passive coping rats when tested for fear responses in an environment where social defeat previously occurred.

Experiment 2: Standard contextual fear testing

Similar to Experiment 1, rats were socially defeated for 7 days. Here, following social defeat we trained rats in a standard contextual fear paradigm (Fig. 2 A) based on Knox et al. 19 . Seven days of social defeat led to a split in defeat latencies with passive coping rats having an average of 265 s ± 17.9 s (N = 21) to social defeat, and active coping rats showing an average of 475 s ± 21.6 s (N = 23) to defeat ( t = 7.38, p < 0.0001; Fig. 2 D). These latencies are in the range of our previous results 5 , 6 , 7 , 8 .

Contextual fear following social defeat stress. ( A ) Experimental design for the testing paradigm utilizing 7 days of social defeat and 3 days of contextual fear testing. ( B – C ). Protocol for contextual fear training ( B ) and extinction and extinction retention testing ( C ), leading to robust fear expression in response to the contextual cue. ( D ) Latencies to social defeat showing splits exhibited by animals tested in this experiment. ( E – H ). Total freezing during conditioning averaged individually across the exploration, shock, ITI, or pre-end portion of the training protocol and assessed by one-way ANOVA followed by Tukey post-hoc tests. ( I – K ). Total freezing expressed as a percent of time spent freezing in 1 min bins over the 8 min testing session for extinction (I, day 9), extinction retention (J, day 10), and persistence of extinction retention (K, day 21). ( L – N ), Repeated measures analyses of freezing responses separated into 1 min bins, with time as the repeated measure. Performance on each day was tested by a two-way repeated measures ANOVA using time and stress group (control, passive coping, and active coping) as factors). * p < 0.05. Control n = 18, passive coping n = 20, active coping n = 22.

Experiment 2.1. Increased baseline freezing in passive coping rats

Data were averaged during each period of contextual fear training on day 8, and freezing was subsequently assessed during exploration (210 s total), shock (5 s total), the inter-trial interval (ITI; 300 s total), and pre-end (60 s total; see timeline in Fig. 2 B). Data were analyzed within each training segment specifically to assess whether there were differences in the amount of freezing that occurred prior to delivery of shocks that persisted past training. Passive coping rats had increased freezing relative to control rats during the exploration phase of training (F 2,39 = 4.169, p = 0.02, Fig. 2 E). There were no differences in other measures (Fig. 2 F,G,H). By the end of the training trial (pre-end period), all rats displayed close to maximum levels of freezing (Fig. 2 H), indicating they had successfully associated the context with fear.

Experiment 2.2. Passive coping rats have reduced extinction retention relative to active coping and control rats

We then examined freezing in the contextual fear chamber during extinction training (day 9), extinction retention (24 h later, day 10), and persistence of extinction retention (11 days following extinction retention testing, day 21). A two-way repeated measures ANOVA showed no differences in freezing during the extinction training (day 9, Fig. 2 I, L). All rats displayed high levels of freezing during this phase, indicating strong contextually conditioned fear behavior. A two-way repeated measures ANOVA on freezing during extinction retention (day 10, Fig. 2 J, M) revealed significant main effects of time (F 7,224 = 5.84, p = 0.001) and group (F 2,32 = 5.281, p = 0.01). There was no interaction effect. Post-hoc tests revealed that passive coping rats had significantly increased freezing relative to non-stressed control rats, while active coping and non-stressed control rats did not differ from each other. These results indicate that passive coping rats had impaired extinction retention relative to active coping rats and non-stressed control rats, but did not show deficits in persistence of extinction retention in the contextual fear paradigm (day 21, Fig. 2 K, N) in the manner they did in the ethological fear paradigm in Experiment 1.

Experiment 3: Analysis of neuronal activity and inter-regional correlations

Following extinction retention testing (day 10), a subset of rats trained in the contextual fear paradigm were analyzed for numbers of c-Fos-expressing cells and inter-region correlations of c-Fos-expressing cells. The density of c-Fos staining in a given region is widely used as a marker for neuronal activation, and inter-region correlations of c-Fos densities may suggest functional connectivity and/or highlight important differences in regionally specific activity between animals with differing levels of oping. Following extinction retention testing (day 10), we examined the density of c-Fos-expressing cells (c-Fos positive cells/total cells) in selected brain regions known to be involved in contextual fear, extinction, and memory (Fig. 3 A, B). We assessed cFos on day 10 as freezing on this day was most strongly differentiated between passively and actively coping rats and control rats.

Analysis of c-Fos in selected brain regions following extinction retention testing. ( A ) Experimental overview of brain regions examined by c-Fos immunohistochemistry following extinction retention testing. ( B ) Regions of interest analyzed within the brains of each subject. Passive coping rats had reduced c-Fos in ( C ) dCA1, dCA3, and DG as well as in ( D ) BLA, although not in ceA. No changes in c-Fos expression were seen in the aPVT, but there was a trend toward lower expression by passive coping animals in pPVT. No differences were observed in the mPFC ( F ) or vHPC ( G ). Data analyzed by one-way ANOVA followed by Tukey’s post-hoc tests on significant results, * p < 0.05, # p < 0.1. CG, anterior cingulate cortex; PL, prelimbic cortex; IL, infralimbic cortex; aPVT, anterior paraventricular thalamus; pPVT, posterior paraventricular thalamus; ceA, central amygdala; BLA, basolateral amygdala; dCA1, dorsal cornu Ammonis-1; dCA3, dorsal cornu Ammonis-3; vCA1, ventral cornu Ammonis-1; vCA3, dorsal cornu Ammonis-3. Scale bar = 200 μm. Control n = 8, passive coping n = 7, active coping n = 9.

Experiment 3.1. Passive coping rats have reduced neuronal activity in the dorsal hippocampus and amygdala during extinction retention testing

Passively coping rats showed lower neuronal activation in hippocampal regions, the BLA, and the posterior PVT compared to actively coping and control rats. Specifically, there was significantly lower activity in dorsal CA1 (dCA1; F 2,20 = 8.29, p = 0.002), dorsal CA3 (dCA3; F 2,19 = 9.627, p = 0.05) and in dentate gyrus (DG; F 2,20 = 19.98, p = 0.0001; Fig. 3 C) of passive coping rats relative to active coping and control rats (Fig. 3 C). Passive coping rats had reduced numbers of c-Fos positive cells in the basolateral amygdala (BLA; F 2,21 = 3.536, p = 0.04; Fig. 3 D), but no significant differences in the central amygdala (ceA). There were no group differences in c-Fos expression in the anterior paraventricular nucleus of the thalamus (aPVT; Fig. 3 E), medial prefrontal cortex (prelimbic, PL; infralimbic, IL), cingulate (CG; Fig. 3 F), or ventral hippocampus CA1 (vCA1) or CA3 (vCA3) regions (Fig. 3 G). There was a trend toward lower c-Fos expression in the posterior paraventricular nucleus of the thalamus (pPVT, F 2,20 = 2.77, p = 0.08). Correlations performed between numbers of c-Fos-expressing cells and percent freezing indicated no significant association between neural activity in any brain region and freezing behavior during extinction retention.

Experiment 3.2. Differences in inter-region correlations of c-Fos within passive coping, active coping, and control populations following extinction retention testing

To assess functional connectivity between brain regions, we first assessed inter-region c-Fos correlations within-subjects (Table 1 ; Radar graphs of all within-subject correlations are presented in Figs. 4 , 5 , 6 ). Control rats had 13 significant correlations, while passive coping and active coping rats had 6 each (see Table 1 ). This difference reveals that net levels of functional connectivity appear to be reduced by stress exposure. Of the significant correlations, passive coping and control rats shared 4 significant correlations, while passive coping and active coping rats shared 2 significant correlations. The only regions that showed significant inter-region correlation in all three groups was the ventral hippocampus (vCA1 vs. vCA3). The only shared significant correlation between control and active coping rats was the positive correlation seen between the PL and dCA1, while passive coping rats did not show such correlated activity between these regions. This could suggest that high functional connectivity between the PL and dCA1 is important for successful fear extinction retention. Control rats had negative correlations between the aPVT and vCA3, pPVT and IL, and pPVT and CG. Neither active coping nor passive coping rats showed any significant correlations with the PVT and other regions. This suggests that a history of stress might reduce the correlation of activity of the PVT with other regions during fear extinction retention testing. Overall, there were greater levels of inter-region correlations in control rats, suggesting that social defeat stress had a broad effect on reducing correlative c-Fos activity across the regions studied, and this could indicate reduced communication between these regions.

Radar graphs made per brain region demonstrating correlations between regions for the dorsal and ventral hippocampus and dentate gyrus. Red lines indicate negative correlations, while blue lines indicate positive correlations. The gray region represents the degree of correlation on an absolute scale presented in r = 0.1 intervals. * p < 0.05.

Radar graphs made per brain region demonstrating correlations between regions for the medial prefrontal cortex. Red lines indicate negative correlations, while blue lines indicate positive correlations. The gray region represents the degree of correlation on an absolute scale presented in r = 0.1 intervals. * p < 0.05.

Radar graphs made per brain region demonstrating correlations between regions for the amygdala and PVT. Red lines indicate negative correlations, while blue lines indicate positive correlations. The gray region represents the degree of correlation on an absolute scale presented in r = 0.1 intervals. * p < 0.05.

Experiment 3.3. Differences in c-Fos correlations between passive coping, active coping, and control rats following extinction testing

To statistically compare correlations in c-Fos densities across pairs of regions between control, passive coping, and active coping populations, we converted Pearson’s r values to z -scores using Fisher’s r to z transformation, which normally-distributes r values and allows statistical comparisons between groups using the z -test (Table 2 ). In the control vs. passive coping comparison, there were 5 significantly different correlation comparisons. In the control vs. active coping comparison, there were 4 significantly different comparisons. There were 5 significantly different comparisons between active coping and passive coping rats. The negative correlation between the pPVT and the IL was significantly different between control rats and both passive coping and active coping rats. This suggests that prior stress exposure prevents the reductions in functional connectivity between the pPVT and IL that occurs in subjects without a prior history of social defeat stress. The significant differences in c-Fos correlations between passive coping and active coping rats all occurred between the amygdala and dorsal hippocampus, and within dorsal hippocampus subregions. Notably, active coping rats, unlike both control and passive coping rats, showed strong correlations between the dCA3 and DG. This suggests that internal circuitry within the dorsal hippocampus during fear extinction retention testing could be an important element of resiliency. An important take away from these results is that while control and active coping rats had similar performance on the contextual fear test, different brain regions were activated during these tests. These results suggests that active coping rats show circuit-based adaptations to the effects of repeated stress that prevent extinction deficits.

The overall goal of this study was to characterize the temporal dynamics of fear extinction and the retention of extinction in stress resilient and vulnerable subpopulations of rats using two separate conditioned fear paradigms: one a naturalistic paradigm employing cues from the cage of a resident aggressor in which the subject was previously defeated in order to evoke fear memories, and the other a standard contextual fear paradigm that uses contextual cues to evoke fear memories. The focus on retention of extinction was informed by both previous studies 19 as well as findings from PTSD patients themselves: individuals with PTSD not only have reduced ability to successfully extinguish fear memory in response to trauma-related cues, but also a reduced ability to successfully reduce fear responses to memories created within experimental settings 20 . Our results reveal that passively coping rats have impaired extinction retention in both paradigms tested, suggesting that they informative for our understanding of PTSD-like symptoms.

In Experiment 1, both passive coping and active coping rats showed increased freezing during extinction training when placed in the cage of the resident that had last defeated them. However, while active coping rats showed reduced freezing 24 h following extinction training (during extinction retention testing), passive coping rats failed to reduce their freezing. This failure to extinguish the fear-associated memory persisted long-term in this ethologically relevant paradigm, as evidenced by increased freezing when tested again within the previously encountered aggressor’s cage 11 days later. Passively coping and actively coping rats were not significantly from each other in extinction retention but passively coping rats exhibited elevated freezing when tested 11 days later showing their impairment in extinction retention persisted. These findings suggest that the underlying circuitry in passive coping rats may prevent the successful retention of a fear extinction memory. In contrast, the circuitry in actively coping rats may underlie why extinction is persistent in some individuals.

In Experiment 2, in which a standard contextual fear conditioning paradigm was used following chronic social defeat, results were largely similar to those in Experiment 1. All groups showed high freezing when tested in the extinction training trial (day 9), perhaps constituting a ceiling effect. However, when tested for extinction retention on day 10, passive coping rats showed increased freezing relative to control rats, but actively coping rats were not different from controls. While we have interpreted this as a failure of these passively coping rats to retain memory for extinction, reductions in freezing across trials of extinction retention during both experiments may instead indicate impairment of extinction re-learning. While the results indicate that actively coping rats are exhibiting reduced freezing during fear learning, it is possible that actively coping rats are displaying passive behaviors that were not captured by our assessments of latencies to be defeated, such as darting or risk assessment. More detailed analyses of behaviors during defeat would be helpful to more fully understand the behavioral strategies used by active and passively coping rats.

In order to determine the neural circuits that could underlie the reduced retention of extinction in passive coping rats, we examined correlations in neuronal activity between regions involved in contextual fear acquisition and extinction following extinction retention testing in the shock-induced contextual fear paradigm as the specific circuits employed in this paradigm have been well-validated 16 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 . Results of the c-Fos analyses showed a reduction in activity in the dorsal hippocampus and BLA of passive coping rats relative to control and active coping rats. This reduction in numbers of c-Fos-expressing cells in the BLA was found in the BLA of passive coping rats, despite these animals showing higher freezing. Traditionally, BLA activation is associated with high freezing, however, this relationship may depend on the specific source of inputs to the BLA 29 , 30 . This suggests that these regions are important for the expression of extinction responses (i.e., less freezing in an extinguished fear-eliciting context) that were observed, and that passive coping rats fail to activate emotionally-salient contextual information important for guiding the expression of extinction. By examining c-Fos profiles and correlations across relevant brain regions, we were then able to infer potential functional connectivity between regions. Since the neural circuits that are important for long-term retention of extinction memories are not well-established, our selection of regions for study was based on fear conditioning-, extinction-, and stress-relevant circuits. Intriguingly, despite low levels of c-Fos activity in the dorsal hippocampus and amygdala, passive coping rats showed high functional connectivity between the dCA3 and both the BLA and CeA. While control and active coping rats had similar levels of freezing during extinction retention testing, they showed marked differences in functional connectivity between the brain regions we assessed. Active coping rats had high levels of connectivity within the dHPC, potentially suggesting a robust activation of contextual representations of the extinction memory, which could drive the display of reduced freezing.

Control rats had more than twice the number of significant inter-region correlations as passive coping or active coping rats. This could indicate that other regions not measured but implicated in previous studies of fear extinction, such as the nucleus accumbens or striatum 31 , 32 might be recruited in passive coping and/or active coping rats to regulate fear expression during extinction retention testing. The only correlation common to both control and active coping rats was the strong positive correlation between the PL and dCA1, which was not observed in passive coping rats. These data suggest that dCA1, which sends projections to the PL through intermediate and/or direct connections 28 , 33 , 34 , could be an important component of the circuit for accessing fear extinction memory.

We examined c-Fos across the anterior–posterior axis of the PVT as neuronal activity in the PVT regulates habituation to repeated stress and responses to novel stress in chronically stressed rats 35 , 36 , 37 , 38 , 39 . One interpretation of reduced freezing following several days of exposure to a fear-evoking stimulus could be explained by habituation, rather than extinction. Neither the anterior nor posterior portion of the PVT showed significant differences in c-Fos staining across groups, although there was a trend toward lower c-Fos expression in the pPVT (as seen in Fig. 3 E). Collectively, these data suggest that differences in freezing were related to extinction-related processes, rather than habituation-related processes. Intriguingly, correlative activity between the PVT and the mPFC differentiated defeated rats from non-defeated control rats suggesting that a prior history of stress alters the engagement of PVT-related circuits in both passive coping and active coping rats.

Successful fear extinction retention involves both acquisition of a fear extinction memory and its retrieval or recall 40 . While there were no obvious differences during extinction acquisition, we could not definitively determine if there was a deficit in the acquisition of fear extinction or in retrieval during retention testing. Results from our functional connectivity analyses, however, could inform future studies aiming to parse out the role of identified circuits in regulating these distinct components of fear extinction retention. The circuits regulating fear extinction and its retention in stressed females are not clear as our study only examined male animals. It is possible that different brain structures are engaged during fear extinction in females and/or the functional connectivity between the same structures identified here is different in females. Taken together, results from this study demonstrate that successful fear extinction in stress resilient rats involves recruitment of new brain circuits during fear extinction retention testing. Indeed, one of the most intriguing findings was that while the behavior of both control and active coping rats is similar, the underlying circuitry recruited to mediate those behaviors is different. These findings suggest that resilience to the effects of stress is produced by recruitment of specific and unique neural circuitry, not just greater or lesser recruitment of circuitry activated in vulnerable individuals or in non-stressed individuals. Importantly, these unique circuits provide a novel target for promoting resiliency.

All experimental procedures were carried out with the approval of the Institutional Animal Care and Use Committee of The Children’s Hospital of Philadelphia Research Institute and in accordance with the NIH guidelines for the care and use of laboratory animals (National Institutes of Health Publication No. 80–23, revised 1996). The methods of the present study have been reported in accordance with ARRIVE guidelines.

Adult male Sprague–Dawley rats (225–250 g) were obtained from Charles River Laboratories. Rats were singly housed under a 12-h light–dark cycle (lights on at 7 am and off at 7 pm) and were given food and water ad libitum. All rats were randomly assigned to groups by a lab member that was not involved with experimental procedures or data analyses. Rats were euthanized by rapid decapitation and their brains were immediately snap-frozen in 2-methylbutane in Experiments 2 and 3.

To assess extinction of fear responses to social defeat, control and socially-defeated rats (passive coping or active coping) were videotaped on day 8 during an 8-min exposure to the empty cage of the resident rat that had defeated them the previous day. On day 7, rats were tested again for freezing in response to the same resident’s cage on day 9 to test for extinction retention and day 20 to test for persistence of extinction retention. Residents remained in their cages in between sessions, and were removed during fear extinction testing to ensure olfactory cues (urine, feces, dander, pheromones, etc.) were available to subject rats. Control rats were exposed to the un-inhabited empty cage they were previously placed in. The experimental design is depicted in Fig. 1 A and B. A trained experimenter blind to group condition measured freezing behavior in the videos. Freezing was defined as the absence of movement, except movement necessary for breathing, for greater than 2 s, and quantified as percentage of total time for each session. The total number of subjects used in this experiment was: non stressed control = 8, passive coping = 7, and active coping = 6.

Experiments 2 and 3: Extinction of contextual fear and c-Fos analyses

The contextual fear paradigm used was previously published 19 and the experimental design is presented in Fig. 2 A–C. The training criterion was that rats display high levels of freezing when re-exposed to the context in which shocks were administered to ensure that large proportions of rats maintained the expression of fear prior to fear extinction retention trials. The contextual fear protocol was as follows: each rat was individually placed in a sound attenuated contextual fear chamber placed in an isolated room (Harvard Apparatus). The floor of each chamber contained stainless steel rods connected to a shock source and grid scrambler that delivered foot shocks as the unconditioned stimulus (US). The chamber contained a low-intensity light and fan, which provided low level background noise. 1% acetic acid was added to the shock grid floor as an olfactory cue. The chamber was cleaned thoroughly between each test subject. Freezing behavior was scored automatically by Packwin software provided with the system and was defined as the absence of movement, except that necessary for breathing, for greater than 2 s, and quantified as percentage of total time for each session.

Contextual fear training occurred 24 h following the 7th and final day of social defeat. Rats were individually placed in the conditioning chamber. The training protocol began with a 210 s period in which subjects were allowed to explore the chamber. Beginning at 211 s, they received five unsignaled footshocks at 1.0 mA, 1 s each, with a 60 s inter-trial interval (ITI). Rats remained in the chamber for 60 s following the last foot shock (pre-end period). On day 9, rats were placed in the same chamber they were trained in for an extinction trial that lasted 8 min. Our criterion for successful learning as assessed during this first extinction trial was that all rats display high levels of freezing during extinction training. The extinction context matched the context rats were trained in (1% acetic acid, fan on, light on). On day 10, rats were tested for retention of extinction using the same contextual cues during an 8 min exposure to the context. On day 21, rats were tested again for persistence of extinction retention.

Three separate cohorts of animals were used in Experiment 2. For c-fos analysis, animals were rapidly decapitated un-anesthetized, and brains were collected and flash-frozen 60 min after the beginning of the 8 min extinction retention trial on day 10. We chose to assess c-Fos after testing for extinction retention, as the specific question of translational relevance was with respect to circuits underlying retention of extinction impairments. Brains were flash frozen in 2-methylbutane and stored at − 80 °C for use in immunohistochemistry and functional connectivity analyses as described below. The total number of subjects used in contextual fear experiments were: non-stressed control n = 18, passive coping n = 20, active coping, n = 22. Of these, the n’s used for c-Fos analyses from Cohort 3 were non-stressed control = 8, passive coping = 7, and active coping = 9.

Immunohistochemistry (IHC) and assessment of functional connectivity

Brains were sectioned in 30 μm sections for IHC. The brain regions examined are presented in Fig. 3 A-B. For the IHC procedure, brain sections were placed in 4% paraformaldehyde for 45 min then sections incubated with the primary antibody for c-Fos (1:1000, Cell Signaling) and using 3,3’-Diaminobenzidine as a chromogen. To analyze images in ImageJ, two researchers blind to the treatment groups counted the number of c-Fos positive cells within a given region of interest. The density of c-Fos-positive cells was averaged over 2–6 sections per animal. Pearson’s r correlation matrix was created using c-Fos profiles between all brain regions. Correlations were converted into Fisher’s z scores using the r to z transformation. This transformation converts r values into a normal distribution, which allows statistical testing between correlations, and z scores of > 1.96 ( i.e., equivalent to p = 0.05) was considered statistically significant. This is a well-validated method for assessing functional activity between brain regions, which we have previously used 41 , 42 , 43 , and a previous study showed that functional connectivity measures using ΔFosB correlations overlaps with manganese-enhanced MRI of neuronal activity 44 . We have published previous work using this method 29 , 42 , 43 . Radar graphs were created for each brain region for additional visualization.

Data analysis and statistical analysis

The average defeat latency for each rat over the course of the 7 days was calculated and entered into an R script used to perform a bootstrap cluster analyses on the average defeat latencies (code available at www.github.com/cookpa/socialdefeat ) as described in Grafe et al. (2018). Latencies are classified as active coping or passive coping based on the probability of resiliency score generated from this analysis that ranges from 0 to 1.0. Active coping animals have probabilities closer to 1.0 while those classified as passive coping have probabilities closer to 0. For statistical comparisons of two groups, we used the Student’s t test and for comparisons of more than two groups, we used an analysis of variance (ANOVA). For contextual fear and ethological fear experiments we used repeated measures ANOVAs with time split into 1 min bins in fear testing as the repeated measure. Significant main and interaction effects were followed by post-hoc tests. An α level of 0.05 (two-tailed) was set for significance. Additional analyses were conducted utilizing one-way ANOVAs on the total percent time freezing over the 8 min training sessions. All statistical analyses were made in SPSS version 17, R, or Prism 8. Rats that did not show a conditioned freezing response at greater than 30% freezing at the first two bins of the first fear extinction session were excluded in analyses. Additionally, any values ±2 standard deviations from a group mean were removed from analyses. Based on these criteria, three animals were removed, and the total Ns are reflected above. All data are represented as means ±SEM.

This manuscript is dedicated to the memory of our colleague and friend Leszek Kubin.

Data availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author [SB].

Yehuda, R. et al. Post-traumatic stress disorder. Nat. Rev. Dis. Primers 1 , 15057 (2015).

Article PubMed Google Scholar

Pitman, R. K. et al. Biological studies of post-traumatic stress disorder. Nat. Rev. Neurosci. 13 , 769–787 (2012).

Article CAS PubMed PubMed Central Google Scholar

Thompson, N. J., Fiorillo, D., Rothbaum, B. O., Ressler, K. J. & Michopoulos, V. Coping strategies as mediators in relation to resilience and posttraumatic stress disorder. J. Affect. Disord. 225 , 153–159 (2018).

Wood, S. K. & Bhatnagar, S. Resilience to the effects of social stress: Evidence from clinical and preclinical studies on the role of coping strategies. Neurobiol. Stress 1 , 164–173 (2015).

Wood, S. K., Walker, H. E., Valentino, R. J. & Bhatnagar, S. Individual differences in reactivity to social stress predict susceptibility and resilience to a depressive phenotype: Role of corticotropin-releasing factor. Endocrinology 151 , 1795–1805 (2010).

Pearson-Leary, J., Eacret, D. & Bhatnagar, S. Interleukin-1α in the ventral hippocampus increases stress vulnerability and inflammation-related processes. Stress 23 , 308–317 (2020).

Article CAS PubMed Google Scholar

Pearson-Leary, J. et al. Inflammation and vascular remodeling in the ventral hippocampus contributes to vulnerability to stress. Transl. Psychiat. 7 , e1160–e1160 (2017).

Article CAS Google Scholar

Pearson-Leary, J. et al. The gut microbiome regulates the increases in depressive-type behaviors and in inflammatory processes in the ventral hippocampus of stress vulnerable rats. Mol. Psychiat. 25 , 1068–1079 (2020).

Article Google Scholar

Chen, R. J. et al. MicroRNAs as biomarkers of resilience or vulnerability to stress. Neuroscience 305 , 36–48 (2015).

Corbett, B. F. et al. Sphingosine-1-phosphate receptor 3 in the medial prefrontal cortex promotes stress resilience by reducing inflammatory processes. Nat. Commun. 10 , 3146 (2019).

Article ADS PubMed PubMed Central Google Scholar

Grafe, L. A., Eacret, D., Dobkin, J. & Bhatnagar, S. Reduced orexin system function contributes to resilience to repeated social stress. eNeuro 5 , 0273 (2018).

Zuj, D. V., Palmer, M. A., Lommen, M. J. J. & Felmingham, K. L. The centrality of fear extinction in linking risk factors to PTSD: A narrative review. Neurosci. Biobehav. Rev. 69 , 15–35 (2016).

Careaga, M. B. L., Girardi, C. E. N. & Suchecki, D. Understanding posttraumatic stress disorder through fear conditioning, extinction and reconsolidation. Neurosci. Biobehav. Rev. 71 , 48–57 (2016).

Garfinkel, S. N. et al. Impaired contextual modulation of memories in PTSD: An fMRI and psychophysiological study of extinction retention and fear renewal. J. Neurosci. 34 , 13435–13443 (2014).

Milad, M. R. et al. Presence and acquired origin of reduced recall for fear extinction in PTSD: Results of a twin study. J. Psychiat. Res. 42 , 515–520 (2008).

Milad, M. R. et al. Neurobiological basis of failure to recall extinction memory in posttraumatic stress disorder. Biol. Psychiat. 66 , 1075–1082 (2009).

Wicking, M. et al. Deficient fear extinction memory in posttraumatic stress disorder. Neurobiol. Learn. Memory 136 , 116–126 (2016).

Liberzon, I. & Abelson, J. L. Context processing and the neurobiology of post-traumatic stress disorder. Neuron 92 , 14–30 (2016).

Knox, D. et al. Single prolonged stress disrupts retention of extinguished fear in rats. Learn. Mem. 19 , 43–49 (2012).

Article PubMed PubMed Central Google Scholar

Tanimizu, T. et al. Functional connectivity of multiple brain regions required for the consolidation of social recognition memory. J. Neurosci. 37 , 4103–4116 (2017).

Lesting, J. et al. Patterns of coupled theta activity in amygdala-hippocampal-prefrontal cortical circuits during fear extinction. PLoS ONE 6 , e21714 (2011).

Article ADS CAS PubMed PubMed Central Google Scholar

Roelofs, K. Freeze for action: neurobiological mechanisms in animal and human freezing. Phil. Trans. R. Soc. B 372 , 20160206 (2017).

Likhtik, E., Popa, D., Apergis-Schoute, J., Fidacaro, G. A. & Paré, D. Amygdala intercalated neurons are required for expression of fear extinction. Nature 454 , 642–645 (2008).

Kreutzmann, J. C., Jovanovic, T. & Fendt, M. Infralimbic cortex activity is required for the expression but not the acquisition of conditioned safety. Psychopharmacology 237 , 2161–2172 (2020).

Laurent, V. & Westbrook, R. F. Inactivation of the infralimbic but not the prelimbic cortex impairs consolidation and retrieval of fear extinction. Learn. Mem. 16 , 520–529 (2009).

Lingawi, N. W., Laurent, V., Westbrook, R. F. & Holmes, N. M. The role of the basolateral amygdala and infralimbic cortex in (re)learning extinction. Psychopharmacology 236 , 303–312 (2019).

Ji, J. & Maren, S. Hippocampal involvement in contextual modulation of fear extinction. Hippocampus 17 , 749–758 (2007).

Ye, X., Kapeller-Libermann, D., Travaglia, A., Inda, M. C. & Alberini, C. M. Direct dorsal hippocampal–prelimbic cortex connections strengthen fear memories. Nat. Neurosci. 20 , 52–61 (2017).

Wiersielis, K. R. et al. Sex differences in corticotropin releasing factor-evoked behavior and activated networks. Psychoneuroendocrinology 73 , 204–216 (2016).

Brockway, E. T., Simon, S. & Drew, M. R. Ventral hippocampal projections to infralimbic cortex and basolateral amygdala are differentially activated by contextual fear and extinction recall. Neurobiol. Learn. Mem. 25 (205), 107832. https://doi.org/10.1016/j.nlm.2023.107832 (2023).

Bouchet, C. A. et al. Activation of nigrostriatal dopamine neurons during fear extinction prevents the renewal of fear. Neuropsychopharmacol. 43 , 665–672 (2018).

Abraham, A. D., Neve, K. A. & Lattal, K. M. Dopamine and extinction: A convergence of theory with fear and reward circuitry. Neurobiol. Learn. Memory 108 , 65–77 (2014).

Nakamura, H., Katayama, Y. & Kawakami, Y. Hippocampal CA1/subiculum-prefrontal cortical pathways induce plastic changes of nociceptive responses in cingulate and prelimbic areas. BMC Neurosci. 11 , 100 (2010).

Jay, T. M., Glowinski, J. & Thierry, A.-M. Selectivity of the hippocampal projection to the prelimbic area of the prefrontal cortex in the rat. Brain Res. 505 , 337–340 (1989).

Bhatnagar, S., Huber, R., Nowak, N. & Trotter, P. Lesions of the posterior paraventricular thalamus block habituation of hypothalamic-pituitary-adrenal responses to repeated restraint: Paraventricular thalamus lesions block habituation to repeated stress. J. Neuroendocrinol. 14 , 403–410 (2002).

Hsu, D. T., Kirouac, G. J., Zubieta, J.-K. & Bhatnagar, S. Contributions of the paraventricular thalamic nucleus in the regulation of stress, motivation, and mood. Front. Behav. Neurosci. 8 , 9956 (2014).

Bhatnagar, S. et al. A cholecystokinin-mediated pathway to the paraventricular thalamus is recruited in chronically stressed rats and regulates hypothalamic-pituitary-adrenal function. J. Neurosci. 20 , 5564–5573 (2000).

Bhatnagar, S. & Dallman, M. Neuroanatomical basis for facilitation of hypothalamic-pituitary-adrenal responses to a novel stressor after chronic stress. Neuroscience 84 , 1025–1039 (1998).

Corbett, B. F. et al. Arc-mediated plasticity in the paraventricular thalamic nucleus promotes habituation to stress. Biol. Psychiat. 5996 , S0006322322000993. https://doi.org/10.1016/j.biopsych.2022.02.012 (2022).

Myers, K. M. & Davis, M. Mechanisms of fear extinction. Mol Psychiat. 12 , 120–150 (2007).

Silva, B. A., Burns, A. M. & Gräff, J. A cFos activation map of remote fear memory attenuation. Psychopharmacology 236 , 369–381 (2019).

Blume, S. R., Nam, H., Luz, S., Bangasser, D. A. & Bhatnagar, S. Sex- and age-dependent effects of orexin 1 receptor blockade on open-field behavior and neuronal activity. Neuroscience 381 , 11–21 (2018).

Salvatore, M. et al. Sex differences in circuits activated by corticotropin releasing factor in rats. Horm. Behav. 97 , 145–153 (2018).

Laine, M. A. et al. Brain activation induced by chronic psychosocial stress in mice. Sci. Rep. 7 , 15061 (2017).

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Acknowledgements

We thank Sabrina Ripsman and Deep Patel for their contributions to their studies.

This work was supported by grant funding from Cohen Veterans Bioscience to Seema Bhatnagar. VEP was supported by 5K00MH126549-05.

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Stress Neurobiology Center, Department of Anesthesiology and Critical Care, Children’s Hospital of Philadelphia Research Institute, Philadelphia, PA, USA

Jiah Pearson-Leary, Valerie Estela-Pro, Elizabeth Feindt-Scott, Jason Yan, Abigail Vigderman, Sandra Luz & Seema Bhatnagar

Department of Biology, Haverford College, Haverford, PA, USA

Alexander P. Abramenko

Department of Anesthesiology and Critical Care, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

Seema Bhatnagar

Center for Behavioral Neuroscience, Neuroscience Institute, Georgia State University, Atlanta, GA, USA

Debra Bangasser

Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

Richard Ross

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Data analysis and manuscript preparation was done by J.P.L. with assistance from A.P.A. and V.E.P.; J.P.L. and S.B. designed the experiments. S.B. edited the manuscript. Data collection was performed by J.P.L. with help from E.F.S., J.Y., A.V., and S.L.; D.B., R.R., and L.K. provided consultation on experimental design and edited the manuscript. All work was performed in the lab of S.B.

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Correspondence to Seema Bhatnagar .

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Pearson-Leary, J., Abramenko, A.P., Estela-Pro, V. et al. Differential recruitment of brain circuits during fear extinction in non-stressed compared to stress resilient animals. Sci Rep 14 , 2125 (2024). https://doi.org/10.1038/s41598-023-50830-w

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Lab Rat Afraid Experiments Stock Photo 527932408
Little Albert Experiment (Watson & Rayner)
Experiment 1: Rats exhibited enhanced conditioned fear extinction
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Shocking Rat Experiment Teaches Powerful Life Lesson

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Observational Fear Learning in Rats: Role of Trait Anxiety and Ultrasonic Vocalization
In this experiment, the rats were first submitted to a light-dark box test (Figure 1 a). Then, the rats were grouped with respect to their prospective roles (DEM, OBS, NAIVE; n = 16/group) in the upcoming observational fear learning experiment with the restriction that there must be two triads with DEM OBS and NAIVE rats per cage.
Cued and Contextual Fear Conditioning for Rodents
Fear conditioning to either a cue or a context represents a form of associative learning that has been well used in many species . The majority of the experiments reported in the literature involve the mouse; however, there is also a generous proportion of the literature devoted to the rat.
Ecological analysis of Pavlovian fear conditioning in rats
The absence of one-trial fear conditioning in a naturalistic setting may be analogous to "The Rat Park Experiment," where rats housed in an enriched environment with plants, trees, and social ...
The complex affective and cognitive capacities of rats
In rats, as in many species, fear has been one of the most researched emotions. It is easily evoked in response to threat or pain and is objectively measured by stress hormones, physiological arousal, freezing, and avoidance responses. ... In another experiment, rats learned to drive a small, motorized vehicle by pressing a lever to move it ...
Using Rats to Trace Anatomy of Fear, Biology of Emotion
Like most animals, rats exhibit fear, an emotion that may help creatures escape from predators. In experiments over the last 15 years, Dr. LeDoux has traced fear inside the rat's brain -- from the ...
Rats sense their human handler's fear
According to a new study, laboratory rats can also detect human fear. To assess whether rats have the ability to detect negative emotional states in humans, the researchers analyzed the behavior ...
Long-lasting incubation of conditioned fear in rats
Exp. 1: Time course of fear incubation. We studied the time course of fear incubation in 4 groups of rats (n=10-15 per group). The rats were trained for fear conditioning over 10 sessions of tone-shock pairings, and then tested for conditioned fear 2, 15, 31, and 61 days (±1 day for days 30 and 60) after the last tone and shock exposure.
Observational Fear Learning in Rats: Role of Trait Anxiety and ...
In the first experiment, trait anxiety was assessed in a light-dark box test before the rats were submitted to the observational fear learning procedure. In the second experiment, ultrasonic vocalization was recorded throughout the whole observational fear learning procedure, and 22 kHz and 50 kHz calls were analyzed.
Observational Fear Learning in Rats: Role of Trait Anxiety and
In the first experiment, trait anxiety was assessed in a light-dark box test before the rats were submitted to the observational fear learning procedure. In the second experiment, ultrasonic vocalization was recorded throughout the whole observational fear learning procedure, and 22 kHz and 50 kHz calls were analyzed.
The conditions that regulate formation of a false fear memory in rats
In four experiments, we investigated how rats learn to fear a context in which they have never experienced danger (i.e., how they form a false context fear memory). In each experiment, rats were pre-exposed to a context on day 1, shocked in a similar-but-different context on day 2, and tested in the pre-exposed or explicitly-conditioned context ...
A fear conditioned cue orchestrates a suite of behaviors in rats
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.
Behavioral and brain mechanisms mediating conditioned flight behavior
As in the previous experiments all rats showed similarly low levels of ... E. A. & McDonald, R. J. Discriminative fear conditioning to context expressed by multiple measures of fear in the rat.
Little Albert experiment
Before the experiment, Albert was given a battery of baseline emotional tests: the infant was exposed, briefly and for the first time, to a white rat, a rabbit, a dog, a monkey, masks (with and without hair), cotton, wool, burning newspapers, and other stimuli. Albert showed no fear of any of these items during the baseline tests.
Little Albert Experiment (Watson & Rayner)
The experiment was performed in 1920 and was a case study aimed at testing the principles of classical conditioning. Watson and Raynor presented Little Albert (a nine-month-old boy) with a white rat, and he showed no fear. Watson then presented the rat with a loud bang that startled Little Albert and made him cry.
Rodent Models of Conditioned Fear: Behavioral Measures of Fear and
In the rat experiments, testing for cued fear memory was conducted in a second chamber novel to the rat subjects. Lighting conditions, box geometry, flooring surface (smooth surface with sawdust bedding as opposed to the conducting rod floor), wall color, and olfactory cues differed from the training chamber. 6.
Behavioral Expression of Contextual Fear in Male and Female Rats
Experiment 1: Freezing. Average time spent freezing during the baseline period and the post-shock period of the fear conditioning session was averaged for each animal. Likewise, the average time spent freezing during the 10 min context test was computed for all rats.
Fearful Memories Passed Down to Mouse Descendants
Fearful Memories Passed Down to Mouse Descendants. From Nature magazine. reports. The authors suggest that a similar phenomenon could influence anxiety and addiction in humans. But some ...
The conditions that regulate formation of a false fear memory in rats
Rudy and colleagues (Rudy & O'Reilly, 1999) used a Pavlovian fear conditioning protocol to demonstrate that animals could become afraid of a context in which an aversive event never occurred.In their experiment, rats that had been pre-exposed to a context would, when shocked in a similar unfamiliar context, show fear to the pre-exposed context even though they had not been shocked there.
Conditioned turning behavior: A Pavlovian fear response expressed
We have previously conducted fear conditioning experiments using a paradigm in which rats are given an auditory CS paired with a unilateral shock US delivered to one eyelid (Moita et al., 2003, 2004; Blair et al., 2005a,b; Tarpley et al. 2009; Johansen et al. 2010). During these experiments, we have observed that in addition to CS-evoked ...
Long-Lasting Incubation of Conditioned Fear in Rats
In 1937, Diven reported that human fear responses to cues previously paired with shock progressively increase or incubate over 24 hours. Since then, fear incubation has been demonstrated in both humans and nonhumans. However, the difficulty of demonstrating long-lasting fear incubation in rodents has hampered the study of the underlying mechanisms of this incubation. Here, we describe a rat ...
Stressed rats fail to exhibit avoidance reactions to innately aversive
Stressed rats exhibited significantly higher levels of freezing than their control counterparts during fear acquisition (Supplementary Fig. 11). 24 h later, when tested for recall of conditioned ...
Evidence for recovery of fear following immediate extinction in rats
Results Humans Reinstatement experiment. Previous studies in our laboratory have developed a human reinstatement paradigm and shown that immediately extinguished conditioned fear can be recovered following reinstatement (LaBar and Phelps 2005).This effect was demonstrated using a single cue delay procedure as well as a delay discrimination procedure, both with a full reinforcement schedule ...
Differential recruitment of brain circuits during fear extinction in
Experiment 1: ethological fear testing. Rats were socially defeated for 7 days (Fig. 1A). During each episode of social stress, a rat was placed into the home cage territory of an unfamiliar Long ...

Using Rats to Trace Anatomy of Fear, Biology of Emotion

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Experiment 1

Experimental design and nose poke suppression.

Representative behaviors.

Behavior during a single danger trial.

Temporal ethograms reveal shifting behavioral patterns over discrimination

Temporal ethograms.

Danger orchestrates a suite of behaviors

Danger-elicited behaviors.

Associatively acquired behaviors generalize early

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Conditioned suppression reveals complete discrimination during extinction with reward apparatus present

Danger-elicited locomotion peaks when foot shock would have occurred

Temporal ethograms during extinction.

Danger elicits locomotion.

Freezing is less dangerspecific and is sensitive to time, test type, and order

Danger-elicited behaviors are independently expressed during extinction

Behavior–behavior correlations during extinction.

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