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Defensive behavioral responses are contingent upon threat intensity, proximity, and context of exposure. Based on these factors, we developed a classical conditioning paradigm that elicits clear transitions between conditioned freezing and flight behavior within individual subjects. This model is crucial for the understanding the pathologies involved in anxiety, panic, and post-traumatic stress disorders.
Fear- and anxiety-related behaviors significantly contribute to an organism’s survival. However, exaggerated defensive responses to perceived threat are characteristic of various anxiety disorders, which are the most prevalent form of mental illness in the United States. Discovering the neurobiological mechanisms responsible for defensive behaviors will aid in the development of novel therapeutic interventions. Pavlovian fear conditioning is a widely used laboratory paradigm to study fear-related learning and memory. A major limitation of traditional Pavlovian fear conditioning paradigms is that freezing is the only defensive behavior monitored. We recently developed a modified Pavlovian fear conditioning paradigm that allows us to study both conditioned freezing and flight (also known as escape) behavior within individual subjects. This model employs higher intensity footshocks and a greater number of pairings between the conditioned stimulus and unconditioned stimulus. Additionally, this conditioned flight paradigm utilizes serial presentation of pure tone and white noise auditory stimuli as the conditioned stimulus. Following conditioning in this paradigm, mice exhibit freezing behavior in response to the tone stimulus, and flight responses during the white noise. This conditioning model can be applied to the study of rapid and flexible transitions between behavioral responses necessary for survival.
Fear is an evolutionarily conserved adaptive response to an immediate threat1,2. While organisms possess innate defensive responses to a threat, learned associations are crucial to elicit appropriate defensive responses to stimuli predictive of danger3. Dysregulation in brain circuits controlling defensive responses is likely to contribute to maladaptive reactions associated with multiple debilitating anxiety disorders, such as post-traumatic stress disorder (PTSD), panic disorder4, and specific phobias5,6. The prevalence rate in the United States for anxiety disorders is 19.1% for adults and 31.9% in adolescents7,8. The burden of these illnesses is extremely high on the daily routine of individuals and negatively impacts their quality of life.
Over the last several decades, Pavlovian fear conditioning has served as a powerful model system to gain tremendous insight into the neural mechanisms underlying fear-related learning and memory9,10,11. Pavlovian fear conditioning entails pairing a conditioned stimulus (CS, such as an auditory stimulus) with an aversive unconditioned stimulus (US; for example, an electrical footshock)12. Because freezing is the dominant behavior evoked and measured in standard Pavlovian conditioning paradigms, the neural control mechanisms of active forms of defensive behavior such as escape/flight responses remain largely unexplored. Previous studies show that different forms of defensive behavior, such as flight, are evoked depending upon the threat intensity, proximity and context13,14. Studying how the brain controls different types of defensive behavior may significantly contribute to the understanding of the neuronal processes that are dysregulated in fear and anxiety disorders.
To address this critical need, we developed a modified Pavlovian conditioning paradigm that elicits flight and escape jumps, in addition to freezing15. In this paradigm, mice are conditioned with a serial compound stimulus (SCS) consisting of a pure tone followed by white noise. Following two days of pairing the SCS with a strong electrical footshock, mice exhibit freezing in response to the tone component and flight during the white noise. Behavioral switches between conditioned freezing and flight behavior are rapid and consistent. Interestingly, mice exhibit flight behavior only when the white noise CS is presented in the same context as a previously delivered footshock (the conditioning context) but not in a neutral context. Instead, freezing responses dominate in this the neutral context, with significantly greater levels of freezing in response to the white noise compared to the tone. This is consistent with the role of context in modulating defensive response intensity and with the regulatory role of contextual information in fear-related learning and memory found in traditional threat conditioning paradigms16,17. This model allows for direct, within-subject comparisons of multiple defensive behaviors in a context-specific manner.
The following steps/procedures were conducted in accordance with institutional guidelines after approval from the Institutional Animal Care & Use Committee of Tulane University.
1. Preparation of mice
2. Preparation of study materials
3. Preparation of computer program and video tracking
4. Behavioral experiment
5. Quantification of behavior
6. Statistical analysis
As described in the diagram (Figure 1A), the session starts with pre-exposure (Day 1), followed by fear conditioning (Days 2 and 3), and then either extinction or retrieval (Day 4).
Presentations of the SCS in the pre-exposure (Day 1) session did not elicit flight or freezing response in the mice (Figure 2A-2B). Behavioral analysis during conditioning (Days 2 and 3) revealed that the tone component of the SCS signific...
The described sound and shock parameters are important elements of this protocol. It is critical, therefore, to test the shock amplitude and sound pressure level before starting the experiments. Fear conditioning studies typically use 70-80 dB sound pressure levels and 0.1-1 mA shock intensity18; thus, the described parameters are within the bounds of traditional fear conditioning paradigms. In a previous CS-only (no footshock) control experiment, we did not observe flight or freezing responses in...
The authors have nothing to disclose.
This work was supported by the Louisiana Board of Regents through the Board of Regents support fund (LEQSF(2018-21)-RD-A-17) and the National Institute of Mental Health of the National Institutes of Health under award number R01MH122561. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Name | Company | Catalog Number | Comments |
Neutral context | Plexiglass cylinder 30 X 30 cm | ||
Fear conditioning box | Med Associates, Inc. | VFC-008 | 25 X 30 X 35 cm dimentions |
Audio generator | Med Associates, Inc. | ANL-926 | |
Shocker | Med Associates Inc. | ENV-414S | Stainless steel grid |
Speaker | Med Associates, Inc. | ENV-224AM | Suitable for pure tone and white noise |
C57/BL6J mice | Jackson laboratory, USA | 664 | Aged 3-5 month |
Cineplex software (Editor/ studio) | Plexon | CinePlex Studio v3.8.0 | For video tracking and behavioral scoring analysis |
MedPC software V | Med Associates, Inc. | SOF-736 | |
Neuroexplorer | Plexon | Used to extract the freezing data scored in PlexonEditor | |
GraphPad Prism 8 | GraphPad Software, Inc. | Version 8 | Statistical analysis software |
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