Name: a novel pavlovian fear conditioning paradigm to study freezing and flight behavior
Uploaded: 2021-01-05T12:30:00
Description: Watch this Scientific Journal Video about A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior at JoVE.com

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.

The following steps/procedures were conducted in accordance with institutional guidelines after approval from the Institutional Animal Care &#38; Use Committee of Tulane University.
1. Preparation of mice
<ol>
	<li>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.</li>
	<li>At least one week before the experiment, house all the mice individua...

<ol>
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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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	
	<tbody>
		<tr >
			<td >Neutral context</td>
			<td ></td>
			<td ></td>
			<td >Plexiglass cylinder 30 X 30 cm&#160;</td>
		</tr>
		<tr >
			<td >Fear conditioning box</td>
			<td >Med Associates, Inc.</td>
			<td >VFC-008</td>
			<td>25 X 30 X 35 cm dimentions</td>
		</tr>
		<tr >
			<td >Audio generator&#160;</td>
			<td >Med Associates, Inc.</td>
			<td >ANL-926&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Shocker</td>
			<td >Med Associates Inc.</td>
			<td >ENV-414S</td>
			<td>Stainless steel grid</td>
		</tr>
		<tr >
			<td >Speaker</td>
			<td >Med Associates, Inc.</td>
			<td >ENV-224AM</td>
			<td>Suitable for pure tone and white noise&#160;</td>
		</tr>
		<tr >
			<td >C57/BL6J mice</td>
			<td >Jackson laboratory, USA</td>
			<td >664</td>
			<td>Aged 3-5 month</td>
		</tr>
		<tr >
			<td >Cineplex software (Editor/ studio)</td>
			<td >Plexon</td>
			<td>CinePlex Studio v3.8.0</td>
			<td>For video tracking and behavioral scoring analysis</td>
		</tr>
		<tr >
			<td >MedPC software V</td>
			<td >Med Associates, Inc.</td>
			<td >SOF-736</td>
			<td></td>
		</tr>
		<tr >
			<td >Neuroexplorer</td>
			<td >Plexon</td>
			<td ></td>
			<td>Used to extract the freezing data scored in PlexonEditor</td>
		</tr>
		<tr >
			<td >GraphPad Prism 8</td>
			<td >GraphPad Software, Inc.</td>
			<td >Version 8</td>
			<td>Statistical analysis software</td>
		</tr>
	</tbody>
</table>

a novel pavlovian fear conditioning paradigm to study freezing and flight behavior

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;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.

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...

Watch this Scientific Journal Video about A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior at JoVE.com

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

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&#8217;s survival. However, exaggerated defensive responses to perceived threat ...

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.

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