This work was funded by National Institute of Health R01AA026530 (MSF), Staglin Center for Brain and Behavioral Health (MSF), NRSA-F32 MH10721201A1 and NARSAD 26612 (AKR), and NSF DGE-1650604 (SG).

1. Subjects
<ol>
	<li>Rats
	<ol>
		<li>Order rats to arrive when they are approximately 90 days old and single-housed in standard rat cages. 
		Note: Single housing is advised, as group housing produces variability due to interactions between animals in the home cage, particularly following stress exposure. SEFL has been demonstrated in male and female rats, in Long-Evans and Sprague Dawley rats, and in rats as young as 19 days old7,10.</li>
		<li>Randomly assign animals to at least two conditions: trauma (n = 8) and no trauma (n = 8) (see Rau et al.5 for additional control conditions).</li>
	</ol>
	</li>
	<li>Mice
	<ol>
		<li>Order mice to arrive when they are approximately 60 days old and single-housed in standard mouse cages. Single-house the mice for at least 4 weeks prior to trauma as well as throughout the experiment duration.</li>
		<li>Randomly assign animals to at least two conditions: trauma (n = 8) and no trauma (n = 8).</li>
	</ol>
	</li>
</ol>
2. Equipment Setup
<ol>
	<li>Set up one set of fear-conditioning chambers to serve as Context A and a second set of fear-conditioning chambers to serve as Context B (see Materials Table). Place each fear-conditioning chamber inside a sound-attenuating cubicle to prevent intrusion of outside noise (see Materials Table).
	<ol>
		<li>Ensure that a method of illuminating the chambers with visible light (such as a white overhead house light) is present in the chambers serving as Context A (see Materials Table).</li>
		<li>Ensure that different solutions are available to clean the chambers between each animal and provide different odors for each chamber (e.g., diluted cleaning solution and 1% acetic acid). 
		Note: It is critical that animal-generated odors be eliminated11.</li>
		<li>Place plastic inserts in Context B to differentiate the internal layout of the two contexts. A black Plexiglas triangular insert is recommended. Alternatively, use a white plastic sheet to create a curved back wall (see Materials Table).</li>
		<li>Place grid floors in each fear conditioning chamber for footshock delivery, using a different grid pattern for each context to differentiate floor texture between contexts (see Materials Table). 
		Note: Sufficiently distinct grid patterns include flat grids (all bars arranged in a single horizontal plane), staggered grids (bars arranged in two offset horizontal planes), and alternating grids (bars arranged in a single horizontal plane but of varying diameters).</li>
		<li>Place clean metal pans beneath each grid floor to collect droppings. Scent pans with the cleaning solution (see Materials Table).</li>
	</ol>
	</li>
	<li>Provide accurate timing and amplitude of footshock delivery for each context.
	<ol>
		<li>Connect a shock generator and scrambler capable of delivering 1 mA or lower amplitude shocks to each grid floor for footshock delivery (see Materials Table). 
		Note: The shock generators and scramblers should be located outside of the sound attenuating chamber, with cables connecting the generators and scrambler to the grid floors via openings in the sound attenuating chamber. This will prevent damage due to chewing, cleaning solution, etc.</li>
		<li>Use a multimeter to test the current being delivered by the shock generator by placing each probe on a different bar of the grid floor and confirming that the desired shock amplitude is produced (see Materials Table).</li>
		<li>Ensure that a method for controlling the timing and amplitude of shock delivery (e.g., computer software) is available (see Materials Table).</li>
	</ol>
	</li>
	<li>Ensure that a method for video-recording each animal during each experimental session is available (see Materials Table). 
	Note: It will be necessary to record both 1) when the chambers are illuminated by visible light and 2) when the chambers are dark. The latter can be accomplished by either using a night vision camera or illuminating and recording the darkened chambers using infrared or near-infrared light.</li>
	<li>Ensure that a distinctive method of transporting animals from the vivarium to Context B is available to further differentiate the two contexts. 
	Note: While methods such as a black plastic tub (38 x 30 x 24 cm) divided into four compartments or clean empty cages have been successfully used, any other transport box that is distinctly different from the home cage can be used.</li>
</ol>
3. SEFL Procedure for Rats and Mice
<ol>
	<li>Handle all rodents daily by gently removing them from the homecage and holding each for 60-90 seconds for at least 7 days before beginning the SEFL procedure.</li>
	<li>On Day 1 of the SEFL procedure, place subjects in Context A, where they will receive the traumatic stressor.
	<ol>
		<li>Set up Context A with one set of grid floors (e.g., flat grids) and illuminate the chambers with visible light.</li>
		<li>Use a multimeter to test the current being delivered by the shock generator by placing each probe on a different bar of the grid floor and confirming that the desired shock amplitude is produced. 
		Note: Damage or corrosion of the bars can result in weak or uneven shock delivery. Liquids including urine touching the grid along the wall can also adversely affect shock delivery.</li>
		<li>Wipe down the chamber walls and doors and spray the pans beneath the grid floors with one solution (e.g., diluted cleaning solution). 
		Note: This is necessary to eliminate odors from the previous animals.</li>
		<li>Transport animals from the vivarium to the experimental room in their home cages placed on a cart and place individually into the fear conditioning chambers. Only bring one round&#8217;s worth of animals (determined by the number of fear conditioning chambers) to the experiment room at a time. 
		Note: To avoid confounds due to order or timing, each round should contain animals in both the trauma and no trauma conditions.</li>
		<li>For rat experiments, use the shock generator and scramblers to deliver 15 1-s, 1-mA footshocks randomly presented over 90 minutes (average ISI = 6 min) through the grid bars of the chambers containing trauma condition subjects. Expose the no trauma controls to the same context for 90 minutes without shock delivery.</li>
		<li>For mouse experiments, use the shock generator and scramblers to deliver 10 1-s, 1-mA footshocks randomly presented over 60 minutes (average ISI = 6 min) through the grid floors of the chambers containing trauma condition subjects. Expose the no trauma controls to the same context for 60 minutes without shock delivery.</li>
		<li>After 90 minutes (rat experiments) or 60 minutes (mouse experiments), return all animals to their homecages and promptly return to the vivarium.</li>
	</ol>
	</li>
	<li>On Day 2 of the SEFL procedure, assess the fear to the trauma context if desired.
	<ol>
		<li>Set up Context A as done on Day 1.</li>
		<li>Transport animals to the experiment room in their home cages as on Day 1.</li>
		<li>Place animals in Context A for 8 minutes without shock delivery and video record the behavior during the entire session.</li>
		<li>After 8 minutes, return all animals to their homecages and promptly return to the vivarium.</li>
	</ol>
	</li>
	<li>On Day 3 of the SEFL procedure, expose all subjects to the mild stressor in Context B. 
	Note: This procedure can occur anywhere from 24 hours to 90 days after the traumatic stressor9.
	<ol>
		<li>Set up Context B with a different set of grid floors from the ones used in Context A (e.g., alternating or staggered grid floors) and black triangular or white curved Plexiglas inserts. Do not illuminate the chambers with visible light; although, infrared or near-infrared light can be used as necessary.</li>
		<li>Use a multimeter to test the current being delivered by the shock generator by placing each probe on a different bar of the grid floor and confirming that the desired shock amplitude is produced. Use a multimeter to test the current being delivered by the shock generator by placing each probe on a different bar of the grid floor and confirming that the desired shock amplitude is produced. 
		Note: Damage or corrosion of the bars can result in weak or uneven shock delivery. Liquids including urine touching the grid along the wall can also adversely affect shock delivery.</li>
		<li>Wipe down the chambers and spray the pans beneath the grid floors with the solution not used in Context A (e.g., 1% acetic acid).</li>
		<li>Transport animals from the vivarium to the experimental room in a method distinct from the method used for Context A (e.g., a black plastic tub) and place them individually into fear conditioning chambers. Only bring one round&#8217;s worth of animals to the experiment room at a time (determined by the number of fear conditioning chambers).</li>
		<li>Expose all animals to the mild stressor (described below) and video record freezing and activity during the session.
		<ol>
			<li>After a 180-s baseline period, deliver either a single 1-s, 1-mA footshock (rats) or a single 2-s, 1-mA footshock (mice) to all animals. 
			Note: Ensure that during the 180-s baseline period freezing should not exceed 5%12.</li>
			<li>Remove all animals 30 seconds after shock delivery and promptly return to vivarium.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>On Day 4 of the SEFL procedure, test fear to the mild stressor context.
	<ol>
		<li>Set up Context B as done on Day 3.</li>
		<li>Transport animals from the vivarium to the experimental room in the same transport as done on Day 3.</li>
		<li>Place animals in Context B for 8 minutes without shock delivery and video record freezing throughout the session.</li>
		<li>Remove all animals after 8 minutes and promptly return to the vivarium.</li>
	</ol>
	</li>
</ol>
4. Data Analysis
<ol>
	<li>Measure fear during the recorded experimental sessions using freezing, defined as the lack of all movement except that which is needed for respiration. 
	Note: Freezing is scored most accurately by a blind human scorer, but there are several automated programs that perform well. However, all automated systems must be calibrated to a human observer to be accurate13.
	<ol>
		<li>To score freezing by hand, have an experimenter blind to experimental conditions observes the subject every 4 seconds throughout the time period of interest3. At each observation, classify the subject as &#8220;freezing&#8221; or &#8220;not freezing&#8221;. Compare the number of freezing observations to the total number of observations to determine the percent time spent freezing.</li>
		<li>To use automated video analysis to score freezing, first verify that the results from automated video analysis match the results obtained from hand-scoring, as a substantially different freezing score from automated analysis may produce inaccurate results. 
		Note: A rat or a mouse that has never been shocked should show freezing between 0 and 5%, while higher values suggest poor calibration of the equipment</li>
	</ol>
	</li>
	<li>Use the methods described above to measure fear during the time periods of interest (described below).
	<ol>
		<li>Measure fear to the trauma context as the percent time spent freezing across the entire 8-min test session on Day 2.</li>
		<li>Measure generalization of fear from the trauma context to the mild stressor context as the percent time spent freezing during the 3-min baseline period in Context B on Day 3 prior to shock delivery. 
		Note: For SEFL, it is important to differentiate the contexts well enough so that there is not substantial generalization.</li>
		<li>Measure fear immediately following the shock on Day 3 as the percent time spent freezing during the 30-s period that follows the shock.</li>
		<li>Measure fear to the mild stressor context as the percent spent time across the entire 8-min test session on Day 4.</li>
	</ol>
	</li>
	<li>Measure shock reactivity by the amount, or velocity, of movement during the 3-s period during and immediately following the shock on Day 3. 
	Note: ANOVAs are recommended for all data analysis, as additional groups (e.g., drug treatment) can be added as necessary.</li>
</ol>

<ol>
	<li>Bremner, J. D., Krystal, J. H., Southwick, S. M., Charney, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+neuroanatomical+correlates+of+the+effects+of+stress+on+memory.">Functional neuroanatomical correlates of the effects of stress on memory.</a> Journal of Traumatic Stress. 8 (4), 527-553 (1995).</li><li>Dykman, R. A., Ackerman, P. T., Newton, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttraumatic+stress+disorder:+a+sensitization+reaction.">Posttraumatic stress disorder: a sensitization reaction.</a> Integrative Physiological and Behavioral Science. 32 (1), 9-18 (1997).</li><li>Fanselow, M. S., Bolles, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naloxone+and+shock-elicited+freezing+in+the+rat.">Naloxone and shock-elicited freezing in the rat.</a> Journal of Comparative and Physiological Psychology. 93 (4), 736-744 (1979).</li><li>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=What+is+Conditioned+Fear?.">What is Conditioned Fear?.</a> Trends in Neurosciences. 7, 460-462 (1984).</li><li>Rau, V., DeCola, J. P., 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=Stress-induced+enhancement+of+fear+learning:+an+animal+model+of+posttraumatic+stress+disorder.">Stress-induced enhancement of fear learning: an animal model of posttraumatic stress disorder.</a> Neuroscience and Biobehavioral Reviews. 29 (8), 1207-1223 (2005).</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>Poulos, A. M., 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=Sensitization+of+fear+learning+to+mild+unconditional+stimuli+in+male+and+female+rats.">Sensitization of fear learning to mild unconditional stimuli in male and female rats.</a> Behavioral Neuroscience. 129 (1), 62-67 (2015).</li><li>Perusini, J. N., 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=Induction+and+Expression+of+Fear+Sensitization+Caused+by+Acute+Traumatic+Stress.">Induction and Expression of Fear Sensitization Caused by Acute Traumatic Stress.</a> Neuropsychopharmacology. 41 (1), 45-57 (2016).</li><li>Rau, V., Fanselow, M. 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G., 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=Automated+assessment+of+pavlovian+conditioned+freezing+and+shock+reactivity+in+mice+using+the+video+freeze+system.">Automated assessment of pavlovian conditioned freezing and shock reactivity in mice using the video freeze system.</a> Frontiers in Behavioral Neuroscience. , (2010).</li><li>Long, V. A., 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=Stress-enhanced+fear+learning+in+rats+is+resistant+to+the+effects+of+immediate+massed+extinction.">Stress-enhanced fear learning in rats is resistant to the effects of immediate massed extinction.</a> Stress. 15 (6), 627-636 (2012).</li><li>Craske, M. G., 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=Optimizing+inhibitory+learning+during+exposure+therapy.">Optimizing inhibitory learning during exposure therapy.</a> Behaviour Research and Therapy. 46 (1), 5-27 (2008).</li><li>Paylor, R., Tracy, R., Wehner, J., Rudy, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DBA/2+and+C57BL/6+mice+differ+in+contextual+fear+but+not+auditory+fear+conditioning.">DBA/2 and C57BL/6 mice differ in contextual fear but not auditory fear conditioning.</a> Behavioral Neuroscience. 108 (4), 810-817 (1994).</li><li>Graham, L. 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Dr. Fanselow is a founding board member of Neurovation Labs.

SEFL is a robust behavioral model of PTSD that can be recapitulated in both rats and mice and can be used to study the sensitized fear responses that characterize PTSD. Following traumatic stress, rodents show an increased fear response in a distinctly different context only after that context is paired with a mild stressor that serves as a reminder of a previous traumatic experience. Following the traumatic stress rodents unsurprisingly show high levels of fear when returned to the traumatic stress context on Day 2, ind...

Fear is a critical behavior for survival, enabling individuals to recognize and respond to threats. However, exaggerated fear responses can contribute to the development of psychiatric disorders such as post-traumatic stress disorder (PTSD). One characteristic of PTSD is an exaggerated response to mild stressors, particularly those reminiscent of the original trauma, and a tendency to develop new fears1,2. In the laboratory, fear is often measured through freezing behavior, which is a reliable and ethologically valid index of fear in humans and rodents3,4. While it is known that PTSD involves dysregulation of fear and enhanced fear expression, there is a lack of robust animal models of PTSD that reliably capture this augmented fear response to a relatively innocuous stimulus.
This protocol provides the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a reliable and robust preclinical model of PTSD, in both rats and mice. SEFL utilizes aspects of Pavlovian fear conditioning, yet it produces distinct responses from normal fear conditioning and recapitulates the enhanced fear following traumatic stress observed in PTSD patients5,6. In this model, a single highly stressful experience (referred to here as trauma) leads to lasting behavioral changes, including extreme fear of stimuli associated with the traumatic event, increased anxiety, increased startle reactivity, and altered glucocorticoid signaling7,8. The major feature of SEFL is that following exposure to a traumatic stressor (a series of unsignaled shocks) in a distinct context, animals show an exaggerated fear response to a mild stressor (e.g., a single shock) in a different context. Importantly, the SEFL effect is not due to the generalization from the trauma context to the novel context or increased shock sensitivity5. In our model, we purposefully utilize procedures that reduce any generalization to a novel context such as distinct transport, odor and grid floor pattern. Therefore, unlike normal fear conditioning, SEFL is a non-associative process that leads to a new fear learning that is disproportionately related to environmental cues not directly associated with the traumatic experience. Extensive work shows that a single 90-minute session containing 15 unpredictable shocks in rats (or a single 60-minute session containing 10 unpredictable shocks in mice) induces a long-lasting sensitization of fear conditioning along with increased anxiety and dysregulation in the circadian rhythm of basal corticosterone. In contrast, pre-exposure to a single footshock does not produce SEFL9. Furthermore, SEFL can be utilized reliably in both rats and mice.
Hence, the SEFL model of PTSD is a powerful tool for probing the biological mechanisms involved in PTSD pathophysiology. Using SEFL, researchers can examine how exposure to a trauma can affect future fear learning. In addition, this model can be useful for investigating specific cellular and molecular mechanisms that may be involved in regulating enhanced fear expression as observed in PTSD.

<table><tbody><tr><td>Fear Conditioning Chamber for Low Profile Floors</td><td>Med Associates Inc.</td><td>VFC-008-LP</td><td>Fear conditioning chamber</td></tr><tr><td>Sound Attenuating Cubicle</td><td>Med Associates Inc.</td><td>NIR-022SD</td><td>Sound-attentuaing cubicle to prevent intrusion of outside noise</td></tr><tr><td>NIR/White Light Control Box</td><td>Med Associates Inc.</td><td>NIR-100VR</td><td>Light control box capable of delivering white and near-infrared light</td></tr><tr><td>NIR VFC Light Box</td><td>Med Associates Inc.</td><td>NIR-100L2</td><td>White overhead houselight</td></tr><tr><td>Windex&amp;nbsp;Original Glass Cleaner</td><td>Windex</td><td></td><td>Solution for cleaning and scenting fear conditioning chambers between animals</td></tr><tr><td>Acetic acid</td><td>Fisher Scientific</td><td>A38-212</td><td>Solution for cleaning and scenting fear conditioning chambers between animals</td></tr><tr><td>A-Frame Chamber Insert</td><td>Med Associates Inc.</td><td>ENV-008-IRT</td><td>Black Plexiglas triangular insert to differentiate internal layout of Contexts A and B</td></tr><tr><td>Curved Wall Insert</td><td>Med Associates Inc.</td><td>VFC-008-CWI</td><td>White plastic sheet to differentiate internal layout of Contexts A and B</td></tr><tr><td>Low Profile Contextual Grid Floor with 1/8&quot; Grid Rods for Mouse</td><td>Med Associates Inc.</td><td>VFC-005A</td><td>Flat grid floor for mice</td></tr><tr><td>Low Profile Contextual Grid Floor with Alternating 1/8&quot; &amp;amp; 3/16&quot; Grid Rods Mouse</td><td>Med Associates Inc.</td><td>VFC-005-S</td><td>Staggered grid floor for mice</td></tr><tr><td>Low Profile Contextual Grid Floor with 1/8&quot; Staggered Grid Rods for Mouse</td><td>Med Associates Inc.</td><td>VFC-005A-L</td><td>Alternating grid floor for mice</td></tr><tr><td>Low Profile Contextual Grid Floor with 3/16&quot; Grid Rods for Rat</td><td>Med Associates Inc.</td><td>VFC-005</td><td>Flat grid floor for rats</td></tr><tr><td>Low Profile Contextual Grid Floor with Alternating 3/16&quot; &amp;amp; 3/8&quot; Grid Rods</td><td>Med Associates Inc.</td><td>VFC-005-L</td><td>Alternating grid floor for rats</td></tr><tr><td>Low Profile Contextual Grid Floor with 3/16&quot; Staggered Grid Rods for Rat</td><td>Med Associates Inc.</td><td>VFC-005-S</td><td>Staggered grid floor for rats</td></tr><tr><td>Metal pans</td><td>Med Associates Inc.</td><td></td><td>Metal pans to catch droppings underneath grid floors</td></tr><tr><td>Standalone Aversive Stimulator/Scrambler</td><td>Med Associates Inc.</td><td>ENV-414S</td><td>Shock generator and scrambler for footshock delivery</td></tr><tr><td>Multimeter</td><td>Fluke</td><td>87-5</td><td>Tool for measuring footshock amplitude</td></tr><tr><td>VideoFreeze Software</td><td>Med Associates Inc.</td><td>SOF-843</td><td>VideoFreeze software for controlling shock delivery</td></tr><tr><td>High Speed Firewire Monochrome Video Camera</td><td>Med Associates Inc.</td><td>VID-CAM-MONO-4</td><td>Video camera capable of recording in near-infrared light</td></tr></tbody></table>

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.

Results of the trauma context test on Day 2 are shown in Figure 1. Animals in the trauma condition showed significantly higher levels of freezing in Context A compared to the no trauma controls, indicating acquisition of fear to the trauma context [rats: F(1,17) = 23.58, p &#60; 0.01; mice: F(1,14) = 666.50, p &#60; 0.0001]. Freezing during the baseline period before the single shock in the novel context on Day 3 is shown in <strong class="x...

Watch this Scientific Journal Video about Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder at JoVE.com

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

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 ...

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11.9K Views.  University of California, Los Angeles. 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. 

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

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Medicine

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 ...

An Inescapable Cat Odor Exposure Protocol for Studying Innate and Contextual Threat Conditioning in Rats

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal model with an ethological approach offers a deeper insight into the biological mechanisms underlying threat responses. This paper describes a methodology to measure defensive behaviors toward both innate and learned aversive stimuli in rats. Animals were individually exposed to predator odor in an inescapable chamber to elicit a measurable, sustained, defensive state. The experimental design involved placing a rat in a familiar chamber for 10 min followed by exposure to cat odor for another 10 min in the same context. The next day, the rats were re-exposed for 10 min to the same context chamber where cat odor exposure occurred. Sessions were video-recorded and defensive behaviors were assessed on both days. 
The behavioral test was coupled with reversible functional inactivation and c-Fos immunohistochemistry techniques to determine the role of the interoceptive cortex in threat responses. Rats exposed to cat odor on the first day and re-exposed to the context chamber on the second day displayed higher levels of defensive behaviors, and that cat odor elicited a robust increase in the neural activity of the interoceptive cortex. Moreover, muscimol inactivation of the interoceptive cortex reduced the expression of defensive behaviors in response to cat odor and impaired contextual threat memory. These results show that this behavioral assay is a useful tool for studying neural mechanisms of defensive behaviors and may offer insight into mechanisms that mediate fear in humans and its related disorders.

Cat skin/fur odor exposure provides a reliable method to investigate neural circuits and mechanisms of defensive responses in rodents and may offer insight into mechanisms that mediate fear in humans. Here, we describe a protocol for investigating the role of the interoceptive cortex in threat responses in rats.

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal ...

A Protocol for the Induction of Posttraumatic Stress-Disorder (PTSD)-like Behavior in Mice

Post-traumatic stress disorder (PTSD) is a debilitating psychiatric condition that precipitates in ~10% of individuals exposed to a traumatic event (TE). Symptoms include recurrent and intrusive thoughts, sleep disturbance, hypervigilance, exaggerated startle, and reckless or destructive behavior. Given the complex and heterogeneous nature of the disease, animal models for PTSD-like symptomatology are of increasing interest to the field of PTSD research. Because resilience to PTSD-like symptomatology is an important epidemiologic aspect of PTSD, animal models that resolve vulnerable and resilient animals are of particular value. Due to the complex nature of the PTSD phenotype and the potential overlaps between PTSD-like behavior and behaviors associated with other stress-induced psychopathologies such as anxiety or depression, animal models that utilize multiple readouts for PTSD-like behavior are also of increasing value. We utilize a paradigm developed by Lebow et al. 2012 for the induction and identification of PTSD-like symptomatology in mice. This paradigm utilizes inescapable electric foot shock, administered in two decontextualized sessions over two consecutive days. Stressed mice perform four behavioral tests - dark/light transfer, marble burying, acoustic startle, and home cage activity - to generate five behavioral readouts of PTSD-like behavior: % risk assessment (%RA), % marbles buried (%MB), % prepulse inhibition (%PPI), latency to peak startle amplitude (LPSA), and % light phase activity (%LPA). PTSD-like symptomatology is characterized by decreased %RA, increased %MB, decreased %PPI, decreased LPSA, and increased %LPA. The 20% of animals displaying the most PTSD-like behavior in each test are awarded a certain number of points depending on the test, and animals scoring sufficient points are designated as PTSD-like, while animals scoring no points are designated PTSD-resilient. This paradigm identifies PTSD-like behavior in ~15% of animals, a rate comparable to that observed in humans. This protocol represents a robust and reproducible paradigm for the induction of PTSD-like behavior in mice.

A Protocol for the Induction of Posttraumatic Stress-Disorder PTSD-like Behavior in Mice

In this study, post-traumatic stress disorder (PTSD)-like behavior is induced in mice using two sessions of inescapable electric foot shock. PTSD-like and resilient animals are identified using several assays for PTSD-specific behaviors.

Post-traumatic stress disorder (PTSD) is a debilitating psychiatric condition that precipitates in ~10% of individuals exposed to a traumatic event (TE). ...

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 ...

Threat-Safety Test for Stress Susceptibility or Resilience in Mice Models

Social Threat-Safety Test Uncovers Psychosocial Stress-Related Phenotypes

Social stress is a major cause of the development of mental disorders. To enhance the translational value of preclinical studies, social stress experience and its behavioral impact on mice should be comparable to humans. Chronic social defeat (CSD) utilizes a type of social stress involving physical attacks and sensory threats to induce mental dysfunctions resembling human affective disorders. To strengthen the psychosocial component of CSD, a 10-day CSD protocol was applied in which daily physical attacks are standardized to three 10 s episodes followed by a 24 h sensory phase. After the 10th sensory phase, the CSD protocol is followed by a refined behavioral assay called the social threat-safety test (STST). Post-stress behavioral assays need to determine how and to what extent the social stressor has influenced behavior. The STST allows chronically socially defeated male mice to interact with 2 novel male individuals (social targets): one social target from the attacking strain encountered during the CSD days and the other from a novel strain. Both are presented simultaneously in different compartments of a three-chambered test arena. The test enables a simultaneous assessment of social avoidance development to measure successful aversive conditioned learning and social threat-safety discrimination ability. The development of social avoidance towards both strains reflects a generalized aversive response and thus, a measurement of stress susceptibility. Meanwhile, the development of social avoidance towards only the attacking strain reflects threat-safety discrimination and thus, a measurement of stress resilience. Finally, the absence of social avoidance towards the attacking strain reflects impaired aversive conditioned learning. The protocol aims to refine the currently used mouse models of stress susceptibility/resilience by including translational criteria, specifically threat-safety discrimination and aversive response generalization, to categorize a single group of chronically socially defeated animals into resilient and susceptible subgroups, eventually advancing future translational approaches.

Author Spotlight: Unveiling Mechanisms of Stress Resilience - Significant Findings, Advancements, and Future Research

The social threat-safety test allows a simultaneous assessment of social avoidance development as a measurement of aversive conditioned learning and social threat-safety discrimination ability, both utilized to identify stress-susceptible and stress-resilient individuals within a single group of chronically socially defeated male mice.

Social stress is a major cause of the development of mental disorders. To enhance the translational value of preclinical studies, social stress experience ...

Modeling Early Life Trauma in Mice to Study PTSD and Social Avoidance Behavior

Chronic Social Defeat Stress in Early Adolescent Male Mice

Physical abuse and trauma in childhood is reported in as many as 1 in 7 children and is a major risk factor for the development of psychiatric diseases, such as Post-Traumatic Stress Disorder (PTSD), in adolescence and adulthood. Hallmark behavioral symptoms of PTSD include avoidance of cues and contexts associated with trauma. The ability to model long-lasting changes in the brain due to early life trauma is critical to determine potential circuit and molecular targets for the modulation of resultant symptoms. This manuscript describes a protocol for modeling early life physical and psychological trauma in early adolescent male mice that produces socially avoidant behavior. Adolescent male mice are exposed to repeated aggressive encounters followed by overnight housing, which provides an added dimension of psychological stress, with an adult male aggressor every day for 10 days. Repeated social defeat paired with overnight housing by the aggressor in early adolescence produces robust social avoidance behavior that lasts throughout adulthood. Social avoidance can be readily quantified in early adolescents, adolescents, and adults using open field social interaction testing. Early adolescent social defeat robustly produces more than 60% susceptible animals in adulthood.

This protocol details how to perform 10 day chronic social defeat stress in early adolescent male mice to produce long-lasting social avoidance in adulthood in more than 60% of mice.

Physical abuse and trauma in childhood is reported in as many as 1 in 7 children and is a major risk factor for the development of psychiatric diseases, ...

Impact of Restraint Stress on Physiology and Neurobiology in Rodent Models

Restraint to Induce Stress in Mice and Rats

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the restraint stress procedure. Restraint stress has been used for decades to investigate changes in physiology, genetics, neurobiology, immunology, and other systems impacted by stress. Due to the ease of performing the procedure, low cost, and numerous modifications to scale for the intensity and duration of stress exposure, a vast literature of studies has used restraint stress in mice and rats. As one example, this study presents previously published data showing the impact of restraint stress in transgenic mice on plasma corticosterone levels and optogenetically-induced norepinephrine release. Acute restraint stress increased plasma corticosterone levels, yet this effect was blunted in mice following repeat restraint stress. However, stimulated norepinephrine release in the bed nucleus of the stria terminalis was increased only in the repeat restraint stress group. These data highlight important considerations of restraint parameters on dependent measures. Additional descriptions of restraint stress in rats are also included for comparison. Finally, the influence of the parameters of the restraint (e.g., acute vs. chronic) and characteristics of the animal subjects (strain, sex, age) are discussed.

This article covers the procedures to induce stress responses using physical restraint stress in mice and rats. Additional considerations that should be observed when selecting and using restraint stress in rodent models are discussed.

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the ...