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

The overall aim of this paradigm is to assess complex defensive behavior in rodents. This protocol is significant because it facilitates investigations into the transitions between defensive behaviors, making it ideal for researchers interested in studying complex adaptive responses to threat. This protocol uses temporarily precise condition stimuli to elicit transition between defensive responses, allowing us to study both, conditioned freezing and flight behaviors within individual subject.

Using this model to uncover the mechanisms of defensive behavior can provide insight into PTSD-related dysfunction and panic disorders and could aid in the development of novel therapeutics. To set up the behavioral protocols, in the program, define the SCS, setting the stimuli to be delivered as either a ten-second pure Tone, or a ten-second White Noise, and defining the inter-trial intervals to be presented pseudo-randomly following each trial. To set up the software tracking, place a test mouse in each relevant context, define the center of gravity, and adjust the contour size.

Use the size of the chambers and the pixel dimensions of the camera to determine the calibration coefficient. Then, generate the TTL pulse code for synchronizing the event markers of the central computer to their real-time occurrences. Before beginning an experiment, turn on the fear-conditioning box controller, shocker, and video recording software.

Mount an overhead speaker above the contexts for delivery of the auditory stimuli at 75 decibels, and set a programmable audio generator to generate auditory stimuli on a predefined schedule. After confirming that the Tone and White Noise are functional, set the system for data acquisition and transport three to five month old male or female mice to the conditioning room. Before beginning the preconditioning trial, clean a 30 centimeter diameter, 30 centimeter high clear cylindrical plexiglass chamber with 1%acetic acid to be used as the Context A chamber.

For preconditioning, place the mouse in Context A and allow it to acclimate for three minutes before exposing the mouse to four 20-second SCS trails with a 90-second average pseudo-random inter-trial interval. After 10 minutes of acclimation, transfer one mouse into the Context A chamber and immediately activate the fear-conditioning system and data collection programs. Before beginning a fear-conditioning experiment, clean an at least 35 centimeter high, 25 by 30 centimeter rectangular enclosure with an electrical grid floor with 70%ethanol to be used as the Context B chamber.

Then, connect the shocker with the electrical grid floor of the Context B chamber, and define the frequency, onset, and duration of the shocks in the appropriate computer program. When all of the parameters have been set, check that the shock intensity is delivered properly from both shocker and grid floor. On days two and three, place the mouse into the Context B chamber for three minutes before exposing the animal to five pairings of the SCS co-terminating with a one-second, 0.9 milliamp AC foot shock and a 120-second average inter-trial interval.

At the end of the session, put the mouse back in the home cage. Depending on the goal of the experiment, to subject the animals to a Recall session, on day four, place the mouse in the Context A chamber for three minutes before presenting the animal with four SCS trials without foot shock with a 90-second average pseudo-random inter-trial interval over 590 seconds to test the animal's Fear Recall response. Or, to test for Fear Extinction, on day four, place the mouse into the Context B chamber for three minutes before subjecting the animal to 16 trials of SCS without foot shock with a 90-second average pseudo-random inter-trial interval over a period of 1, 910 seconds.

To quantify the mouse's behavior, at the end of the experiment, have an observer blind to the analysis score the recorded videos for freezing behavior using automatic freezing detector thresholding followed by a frame-by-frame analysis of the pixel changes. Define freezing as a complete cessation of bodily movements, except for those required for respiration for a minimum of one second. Score a jump as an instance when all four paws leave the floor, resulting in a vertical and/or horizontal movement.

When the entire segment has been analyzed, export the marked file with freezing, jump and event markers and extract the relevant events from the defined time periods of interest into a spreadsheet. To calculate the duration of freezing, subtract the start time from the end time for each respective trial period. Sum the total number of jumps from a particular trial duration.

To calculate the speed of the mouse, track the coordinates from the frame-by-frame XY axis movement of the center of gravity of the mouse. To calculate the flight scores, divide the average speed during each SCS by the average speed during the ten-second pre-SCS, and add one point for each escape jump. A flight score of one indicates no change in flight behavior from the pre-SCS period.

Then, analyze the data for statistical significance using an appropriate statistical analysis software program. SCS presentations in the pre-exposure session do not elicit flight or freezing responses in mice. Behavioral analysis during conditioning reveals that the Tone component of the SCS significantly enhances freezing compared to freezing during the pre-SCS.

Flight scores changed significantly across sessions, and mice exhibit higher speeds and more jumps to the White Noise cue compared to the Tone cue. Mice show a clear defensive behavior transition, exhibiting lower flight scores during the Tone and higher flight scores during the White Noise, with the opposite observed for freezing responses. Mice subjected to 16 trials of extinction training demonstrate a rapid extinction of conditioned flight, with fight scores during the first block of four trials measuring higher during White Noise compared to the Tone cue.

At the end of the extinction session, flight behavior is no longer elicited by either cue. Freezing to the Tone is significantly higher than freezing to the White Noise for the first block of four trials during extinction. Tone-induced freezing decreases over the 16 trials of extinction, while an increase in White Noise-mediated freezing is observed.

In the Recall session, exposure to White Noise in a neutral context does not elicit flight, as the flight scores are below one. Rather, White Noise presentations in the neutral context elicit freezing responses that are higher than those elicited by the Tone. It is important to clean the context throughoutly, and it is critical to test the shock amplitude and sound pressure level before starting an experiment.

This paradigm is currently being used by groups who are interested in understanding the complexities of defensive behavior, and can be used to further our understanding of defensive action selection.