A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior

Summary

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.

Abstract

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.

Introduction

Fear is an evolutionarily conserved adaptive response to an immediate threat^1,2. 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 phobias^5,6

Protocol

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

1. Use male and/or female adult mice aged between 3-5 months. In the present study, we used male C57BL/6J mice obtained from Jackson Laboratory, but any mouse strain from a reputable supplier can be used.
2. At least one week before the experiment, house all the mice individually on a 12:12 h light/dark cycle throughout the study. Provide the mice ad libitum access to food and water.
3. Perform all behaviora....

Results

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.......

Discussion

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.......

Materials

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