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 individually on a 12:12 h light/dark cycle throughout the study. Provide the mice ad libitum access to food and water.</li>
	<li>Perform all behavioral experiments during the light cycle. Perform all sessions at the same time of day within an individual cohort. For example, if starting the experiment at 9 AM on Day 1, continue starting at that time until the experiment is completed.</li>
</ol>
2. Preparation of study materials
<ol>
	<li>Study contexts
	<ol>
		<li>Choose two different contexts to perform the experiments in.</li>
		<li>Use a cylindrical chamber composed of clear Plexiglas (diameter 30 cm) as context A, with a smooth Plexiglas floor. The height of the chamber should be sufficient to prevent escape (at least 30 cm high).</li>
		<li>For Context B use a rectangular enclosure (25 cm x 30 cm) with an electrical grid floor used to deliver alternating current footshocks. The height of this chamber is very important and should be at least 35 cm. high. Alternatively, use a transparent roof (ensure that video can be recorded through this material). 
		NOTE: Use a chamber with smooth wall surfaces that can be easily cleaned.</li>
		<li>Use a different cleaning solution to clean the contexts. For example, clean context A with 1% acetic acid and context B with 70% ethanol. Clean the contexts before beginning the first session, between testing individual mice, and after completion of the day&#8217;s sessions. This is vital to remove the olfactory cues from previous mice. Thorough cleaning will also help prevent urine scaling on the shock grid, which will compromise conditioning sessions. 
		NOTE: The cleaning solutions also serve as an olfactory cue, therefore use the same cleaning liquid for a particular context.</li>
		<li>Place context A or context B in a sound-attenuating box during respective study sessions.</li>
	</ol>
	</li>
	<li>Audio generator
	<ol>
		<li>Mount an overhead speaker above the contexts to deliver auditory stimuli at 75 dB.</li>
		<li>Use a programmable audio generator to generate auditory stimuli on a pre-defined schedule. The 7.5 kHz pure tone is a sound with a sinusoidal waveform, whereas the white noise is a random signal having equal intensity at different frequencies, ranging from 1-20,000 Hz.</li>
		<li>Use TTL pulses to deliver auditory stimuli and shock signals with temporal precision. 
		NOTE: Before starting the experiments, measure the sound intensity output from the mounted speaker in each chamber using a dB meter.</li>
	</ol>
	</li>
	<li>Shocker: Connect the shocker with the electrical grid floor which is used to deliver the 0.9 mA AC shock. Define the frequency, onset, and duration of shocks in a computer program. Deliver each shock stimulus at the end of each SCS for a duration of 1 s, totaling five SCS-shock pairings per conditioning session.</li>
</ol>
3. Preparation of computer program and video tracking
<ol>
	<li>Generate behavioral protocols using coding in a software program.</li>
	<li>In the program, define the serial compound stimulus SCS. This stimulus is a serial presentation of a 10 s pure tone (each pip is presented for 500 ms, at frequency of 7.5 kHz and rate of 1 Hz) and 10 s white noise (500 ms pips at 1 Hz).</li>
	<li>Define the inter-trial intervals (ITI) presented following each trial, pseudorandomly.</li>
	<li>During the study, record all mouse behavior to video for subsequent analysis. 
	NOTE: Commercially available fear conditioning boxes may not be set up to record the behaviors through the top-mounted camera. This is very important since the recorded video is used to calculate horizontal&#160;movement, speed and total distance travelled by the animal.</li>
	<li>For setting the software tracking, place a test mouse in each relevant context, adjust the contour tracking sensitivity, and define the center of gravity. This will ensure the acquisition of reliable data on relative position. In addition, define the whole context area accessible to the subject. 
	NOTE: The adjustment of contour size for both contexts is important as the change in brightness in different contexts will change the contour size.</li>
	<li>Determine a calibration coefficient using the chambers&#8217; known sizes and the camera&#8217;s pixel dimensions which can be used to&#160;calculate speed (cm/s).</li>
	<li>Synchronize the data acquisition computer&#8217;s timestamp events to their real-time occurrences.</li>
</ol>
4. Behavioral experiment
<ol>
	<li>Turn on all the equipment: computers, fear conditioning box controller, shocker, and video and timestamp recording software. Make sure instruments are turned on in the appropriate order.</li>
	<li>Check all the functions including tone, white noise, and shock delivery, and set up the system for the data acquisition.</li>
	<li>Transport the animals from their storage room to the conditioning room. Allow them to acclimatize there for at least 10 min.</li>
	<li>Take the animal out from the home cage, gently place it in the respective context, and then immediately activate the computer programs. 
	NOTE: The initialization of both fear conditioning system and data collection (timestamps, mouse tracking and video recording) software at a time can be synchronized using TTL pulse mediated activations.</li>
	<li>Pre-conditioning/Pre-exposure
	<ol>
		<li>On Day 1, place the subject into context A (neutral context). Allow it to acclimate to the chamber for 3 min (the baseline period), and then expose it to 4 trials of a SCS of 20 s total duration (Figure 1A-1B).</li>
		<li>Maintain a&#160;90 s average pseudorandom&#160;ITI&#160;(range 80-100 s). The total duration of each pre-exposure session is 590 s.</li>
	</ol>
	</li>
	<li>Fear conditioning
	<ol>
		<li>On Day 2 and Day 3, place the subject into Context B. Following a 3 min baseline period, expose the subject to five pairings of the SCS co-terminating with a 1 s, 0.9 mA AC footshock.</li>
		<li>Maintain a 120 s average pseudorandom ITI (range 90-150 s). Have each conditioning session last for 820 s in total (Figure 1A).</li>
		<li>Depending on the goal of the experiment, subject mice on Day 4 to either a recall test (see step 4.7) or to fear extinction (see step 4.8).</li>
	</ol>
	</li>
	<li>Fear recall (to test context dependence)
	<ol>
		<li>On Day 4, place the subject into Context A. After the 3 min baseline period, present it with 4 trials of the SCS without footshock, over 590 s.</li>
		<li>Maintain a 90 s average pseudorandom ITI (range 80-100 s).</li>
	</ol>
	</li>
	<li>Fear extinction
	<ol>
		<li>On Day 4, place the subject into context B. Following the 3 min baseline period, present 16 trials of the SCS without footshock, over 1910 s.</li>
		<li>Maintain a 90 s average pseudorandom ITI (range 60-120 s).</li>
	</ol>
	</li>
	<li>Return the animal to its home cage and repeat the procedure for all the animals.</li>
</ol>
5. Quantification of behavior
<ol>
	<li>Have an observer blind to the experiment score the recorded videos for freezing behavior using automatic freezing detector thresholding followed by a frame-by-frame analysis of pixel changes. 
	NOTE: Other software packages can also be used to calculate freezing automatically by using 2 camera system. It is also possible for an observer to manually score freezing behavior.</li>
	<li>Define freezing as a complete cessation of bodily movements, except for those required for respiration, for a minimum of 1 s.</li>
	<li>Score jumps when all 4 of the paws leave the floor, resulting in a vertical and/or horizontal movement.</li>
	<li>Export the marked file with freezing, jump and event markers.</li>
	<li>Extract relevant events (freezing and jumps) from defined time periods (e.g., 10 s duration of pre-SCS, tone and white noise, for each trial).</li>
	<li>Using the extracted start-stop durations of events in a spreadsheet file, calculate the duration of freezing (in s) by subtracting start time from end time, from the respective trial periods.</li>
	<li>Represent this data trial-wise or day-wise, by summing up freezing duration from all trials. 
	NOTE: Depending on the purpose of the study, the flight or freezing behaviors can be scored and calculated from any trial/duration from the study session.</li>
	<li>Sum the total number of jumps from a particular trial duration.</li>
	<li>Extract the file generated by mouse tracking coordinates from frame by frame X-Y axis movement of the center of gravity of mouse and calculate the speed of the mouse (cm/s). 
	NOTE: The speed data may be present either in the cm/s or pixel/s format. Convert the pixel/s unit to cm/s by using inch or cm/pixel value defined in the video for that testing context (please see section 3.6).</li>
	<li>After extracting speed data for frame by frame movement of the animal, based on frame rate of the video (preferably 30 frames/s), calculate the average speed of the animal in a specific frame number bracket (multiply start and end times in s by 30 to get the start and end frame number).</li>
	<li>Calculate the flight scores by dividing the average speed during each SCS by the average speed during the 10 s pre-SCS (baseline, BL) and then adding 1 point for each escape jump (speedCS/speedBL + # of jumps). A flight score of 1 therefore indicates no change in flight behavior from the pre-SCS period.</li>
	<li>Optionally, score videos manually for other behaviors such as rearing and grooming.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Analyze data for statistical significance using statistical analysis software. For all tests, the definition of statistical significance is P&#60;0.05.</li>
	<li>Check the data for normal distribution using the Shapiro-Wilk normality test (&#945;=0.05).</li>
	<li>To test the effect of cues, carry out the pairwise comparisons using the appropriate parametric (paired t-test) or non-parametric (Wilcoxon signed-rank test) test.</li>
	<li>To assess the 2-way interaction of factors (cue X trial), perform a 2-way ANOVA followed by post-hoc tests (e.g., Bonferroni&#8217;s multiple comparison test/Tukey&#8217;s test).</li>
</ol>

<ol>
	<li>Gross, C. T., Canteras, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+paths+to+fear.">The many paths to fear.</a> Nature Reviews Neuroscience. 13 (9), 651-658 (2012).</li><li>LeDoux, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rethinking+the+Emotional+Brain.">Rethinking the Emotional Brain.</a> Neuron. , (2012).</li><li>Maren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+Pavlovian+fear+conditioning.">Neurobiology of Pavlovian fear conditioning.</a> Annual Review of Neuroscience. 24, 897-931 (2001).</li><li>Johnson, P. L., Truitt, W. A., Fitz, S. D., Lowry, C. A., Shekhar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+pathways+underlying+lactate-induced+panic.">Neural pathways underlying lactate-induced panic.</a> Neuropsychopharmacology. 33 (9), 2093-2107 (2008).</li><li>Mobbs, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+threat+to+fear:+The+neural+organization+of+defensive+fear+systems+in+humans.">From threat to fear: The neural organization of defensive fear systems in humans.</a> Journal of Neuroscience. 29 (39), 12236-12243 (2009).</li><li>M&#252;nsterk&#246;tter, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+or+no+spider?+neural+correlates+of+sustained+and+phasic+fear+in+spider+phobia.">Spider or no spider? neural correlates of sustained and phasic fear in spider phobia.</a> Depression and Anxiety. 32 (9), 656-663 (2015).</li><li>Kessler, R. C., Wai, T. C., Demler, O., Walters, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence,+severity,+and+comorbidity+of+12-month+DSM-IV+disorders+in+the+National+Comorbidity+Survey+Replication.">Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication.</a> Archives of General Psychiatry. 62 (6), 617-627 (2005).</li><li>National Institute of Mental Health. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+anxiety+disorder.">Generalized anxiety disorder.</a> National Institute of Mental Health. , 3-8 (2017).</li><li>Herry, C., Johansen, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encoding+of+fear+learning+and+memory+in+distributed+neuronal+circuits.">Encoding of fear learning and memory in distributed neuronal circuits.</a> Nature Neuroscience. 17 (12), 1644-1654 (2014).</li><li>Janak, P. H., Tye, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+circuits+to+behaviour+in+the+amygdala.">From circuits to behaviour in the amygdala.</a> Nature. 517 (7534), 284-292 (2015).</li><li>Tovote, P., Fadok, J. P., L&#252;thi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+circuits+for+fear+and+anxiety.">Neuronal circuits for fear and anxiety.</a> Nature Reviews Neuroscience. 16 (6), 317-331 (2015).</li><li>Seidenbecher, T., Laxmi, T. R., Stork, O., Pape, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amygdalar+and+hippocampal+theta+rhythm+synchronization+during+fear+memory+retrieval.">Amygdalar and hippocampal theta rhythm synchronization during fear memory retrieval.</a> Science. 301 (5634), 846-850 (2003).</li><li>Blanchard, D. C., Blanchard, R. J., Blanchard, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defensive+behaviors,+fear,+and+anxiety.">Defensive behaviors, fear, and anxiety.</a> Handbook of Anxiety and Fear. Handbook of behavioral neuroscience. , 63-79 (2008).</li><li>Perusini, J. N., Fanselow, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobehavioral+perspectives+on+the+distinction+between+fear+and+anxiety.">Neurobehavioral perspectives on the distinction between fear and anxiety.</a> Learning and Memory. 22 (9), 417-425 (2015).</li><li>Fadok, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+competitive+inhibitory+circuit+for+selection+of+active+and+passive+fear+responses.">A competitive inhibitory circuit for selection of active and passive fear responses.</a> Nature. 542 (7639), 96-99 (2017).</li><li>Maren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+or+electrolytic+lesions+of+the+ventral+subiculum+produce+deficits+in+the+acquisition+and+expression+of+Pavlovian+fear+conditioning+in+rats.">Neurotoxic or electrolytic lesions of the ventral subiculum produce deficits in the acquisition and expression of Pavlovian fear conditioning in rats.</a> Behavioral Neuroscience. 113 (2), 283-290 (1999).</li><li>Xu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+hippocampal+pathways+mediate+dissociable+roles+of+context+in+memory+retrieval.">Distinct hippocampal pathways mediate dissociable roles of context in memory retrieval.</a> Cell. 167 (4), 961-972 (2016).</li><li>Curzon, P., Rustay, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2:+Cued+and+contextual+fear+conditioning+for+rodents.">Chapter 2: Cued and contextual fear conditioning for rodents.</a> Methods of Behavior Analysis in Neuroscience. 2nd edition. , (2009).</li><li>Mollenauer, S., Bryson, R., Robison, M., Phillips, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+avoidance+in+the+C57BL/6J+mouse.">Noise avoidance in the C57BL/6J mouse.</a> Animal Learning &amp; Behavior. 20 (1), 25-32 (1992).</li><li>Hersman, S., Allen, D., Hashimoto, M., Brito, S. I., Anthony, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus+salience+determines+defensive+behaviors+elicited+by+aversively+conditioned+serial+compound+auditory+stimuli.">Stimulus salience determines defensive behaviors elicited by aversively conditioned serial compound auditory stimuli.</a> eLife. 9, (2020).</li><li>Dong, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+cortico-intrathalamic+circuit+for+flight+behavior.">A novel cortico-intrathalamic circuit for flight behavior.</a> Nature Neuroscience. 22 (6), 941-949 (2019).</li><li>Borkar, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+behavioral+responses+during+a+conditioned+flight+paradigm.">Sex differences in behavioral responses during a conditioned flight paradigm.</a> Behavioural Brain Research. 389, 112623 (2020).</li><li>Fadok, J. P., Markovic, M., Tovote, P., L&#252;thi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+perspectives+on+central+amygdala+function.">New perspectives on central amygdala function.</a> Current Opinion in Neurobiology. 49, 141-147 (2018).</li><li>Pitman, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+studies+of+post-traumatic+stress+disorder.">Biological studies of post-traumatic stress disorder.</a> Nature Reviews Neuroscience. 13 (11), 769-787 (2012).</li><li>Canteras, N. S., Graeff, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Executive+and+modulatory+neural+circuits+of+defensive+reactions:+Implications+for+panic+disorder.">Executive and modulatory neural circuits of defensive reactions: Implications for panic disorder.</a> Neuroscience and Biobehavioral Reviews. , (2014).</li></ol>

The authors have nothing to disclose.

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><tbody><tr><td>Neutral context</td><td></td><td></td><td>Plexiglass cylinder 30 X 30 cm&amp;nbsp;</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&amp;nbsp;</td><td>Med Associates, Inc.</td><td>ANL-926&amp;nbsp;</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&amp;nbsp;</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 ...

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6.6K Views.  Tulane University. 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.

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

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e., &#8220;startle probes&#8221;). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g., neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.

Potentiation of the startle reflex is measured via electromyography of the orbicularis oculi muscle during low (uncertain) and high (certain) probability electric shock threat in the Threat Probability Task. This provides an objective measure of distinct negative emotional states (fear/anxiety) for research on psychopathology, substance use/abuse, and broad affective science.

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2 . Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4.
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16, although this may vary depending on the strength and age of the memory15. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e., extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16, episodic17, or declarative18 memories leads to interference with the original memory trace14. We outline below a novel protocol used to block fear recovery in humans.

Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

Neuroscience

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders ...

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the relationships between aversive events and otherwise neutral stimuli. The most-widely utilized procedures for studying emotional memories entail fear conditioning in rats. In these tasks, the unconditioned stimulus (US) is a footshock presented once or several times across single or several sessions, and the conditioned response (CR) is freezing. In a version of these procedures, called cued fear conditioning, a tone (conditioned stimulus, CS) is paired with footshocks (US) during the training phase. During the first test, animals are exposed to the same context in which training took place, and freezing responses are tested in the absence of footshocks and tones (i.e., a context test). During the second test, freezing is measured when the context is changed (e.g., by manipulating the smell and walls of the experimental chamber) and the tone is presented in the absence of footshocks (i.e., a cue test). Most cued fear conditioning procedures entail few tone-shock pairings (e.g., 1-3 trials in a single session). There is a growing interest in less common versions involving an extensive number of pairings (i.e., overtraining) related to the long-lasting effect called fear incubation (i.e., fear responses increase over time without further exposure to aversive events or conditioned stimuli). Extended fear-conditioning tasks have been key to the understanding of fear incubation&#8217;s behavioral and neurobiological aspects, including its relationship with other psychological phenomena (e.g., post-traumatic stress disorder). Here, we describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

We describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the ...

Studying Active Defensive Behaviors in Mice with Modified Fear Conditioning

Modified Fear Conditioning for Inducing Flight Behaviors in Mice

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active defensive behavior, such as jumping or rapid darting, is expressed under high threat imminence or actual threat, whereas passive defensive behavior, such as freezing, is expressed when the threat is predicted, but the threat imminence is relatively low. In classical fear conditioning, subjects typically exhibit freezing as a conditioned defensive response, with little expression of active defensive behavior in most cases. Here, we introduce a modified fear conditioning procedure for mice to observe the transition from freezing to flight and vice versa, involving five repetitive pairings of conditioned stimuli (CS; continuous tone, 8 kHz, 95 dB SPL (sound pressure levels)) and unconditioned stimuli (US; foot shock, 0.9 mA, 1.0 s) over two days. This modified fear conditioning procedure requires a relatively large number of conditioning sessions and conditioning days but does not necessitate a high-intensity foot shock for modest expression of flight behavior. Using the same context for conditioning and salient CS presentations is essential to elicit flight behaviors. This modified fear conditioning procedure is a reliable method for observing active defensive behaviors in mice, providing an opportunity to elucidate the fine mechanisms and characteristics of such behaviors in a fearful context.

Author Spotlight: Exploring Neural Correlates of Defensive Behaviors in Fear Learning and Extinction

To study flight behaviors in a fearful context, we introduce a modified fear conditioning protocol. This protocol ensures that mice consistently exhibit flight behaviors during cue presentation in the fear conditioning.

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active ...

Assessing Rodent Empathy: Observational Fear and Vicarious Freezing Method

Observational Fear as a Model of Affective Empathy in Mice

Empathy, characterized by the ability to recognize and share the emotions of others, plays a fundamental role in shaping social interactions. It allows individuals to respond to the emotional states of others, promoting prosocial behaviors and social bonding. Observational fear is a fundamental aspect of affective empathy, where an observer witnesses a demonstrator undergoing aversive experiences and subsequently exhibits fear behaviors. This socially induced vicarious freezing response in observers, known as emotional contagion or affect sharing, is regarded as an indicator of empathy-like traits in rodents. Here, we present a protocol that delineates the assessment of vicarious freezing responses in observers exposed to conspecific demonstrators receiving aversive foot shocks. The utilization of observational fear assays in rodents has become a widely adopted method for studying the neural mechanisms underlying affective empathy. Given the universality of observational fear across mammals, this methodology further contributes to advancing our understanding of the neural substrates of empathy in humans.

The observational fear paradigm assesses vicarious freezing in rodents as a model for affective empathy. This procedure entails exposing observer rodents to conspecific demonstrators receiving aversive foot shocks to elicit empathic freezing responses. By employing observational fear assays, researchers can investigate the neural mechanisms underlying affective empathy.

Empathy, characterized by the ability to recognize and share the emotions of others, plays a fundamental role in shaping social interactions. It allows ...

Fear Conditioning

Fear Conditioning is a type of learning in which an association is established between a negative unpleasant event and a harmless stimulus. This leads to a fear of the harmless stimulus. This process is largely mediated by the amygdala, which is a brain region involved in emotions and stress reactions. Fear conditioning can be utilized in several ways to understand different aspects of learning and memory.
This video presents an overview of the principles behind fear conditioning, discusses the equipment and a generalized procedure used for this type of experiment. Finally, we'll review some real world applications of fear conditioning in behavioral neuroscience research today.

Fear Conditioning- Classical Conditioning: Principle and Procedure

Fear Conditioning is a type of learning in which an association is established between a negative unpleasant event and a harmless stimulus. This leads to ...