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...

<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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+a+stressor+produces+a+long+lasting+enhancement+of+fear+learning+in+rats.">Exposure to a stressor produces a long lasting enhancement of fear learning in rats.</a> Stress. 12 (2), 125-133 (2009).</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=Amnesia+for+early+life+stress+does+not+preclude+the+adult+development+of+posttraumatic+stress+disorder+symptoms+in+rats.">Amnesia for early life stress does not preclude the adult development of posttraumatic stress disorder symptoms in rats.</a> Biological Psychiatry. 76 (4), 306-314 (2014).</li><li>Fanselow, M. S., Sigmundi, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-specific+danger+signals,+endogenous+opioid+analgesia,+and+defensive+behavior.">Species-specific danger signals, endogenous opioid analgesia, and defensive behavior.</a> Journal of Experimental Psychology: Animal Behavior Processes. 12 (3), 301-309 (1986).</li><li>Jacobs, N. S., Cushman, J. D., 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=The+accurate+measurement+of+fear+memory+in+Pavlovian+conditioning:+Resolving+the+baseline+issue.">The accurate measurement of fear memory in Pavlovian conditioning: Resolving the baseline issue.</a> Journal of Neuroscience Methods. 190 (2), 235-239 (2010).</li><li>Anagnostaras, S. 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. 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=Strain+and+sex+differences+in+fear+conditioning:+22+kHz+ultrasonic+vocalizations+and+freezing+in+rats.">Strain and sex differences in fear conditioning: 22 kHz ultrasonic vocalizations and freezing in rats.</a> Pyschology and Neuroscience. 2 (2), 219-225 (2009).</li></ol>

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&#160;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&#34; 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&#34; &#38; 3/16&#34; 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&#34; 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&#34; 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&#34; &#38; 3/8&#34; 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&#34; 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 ...

This method can help answer key questions in the behavioral neuroscience field, such as how to better understand underlying biological mechanisms of PTSD. The main advantage of this technique is it is robust, reliable, quantitative, and can be performed in multiple species. Begin by setting 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.Place each fear conditioning chamber inside a sound attenuating cubicle to prevent intrusion of outside noise.Then place grid floors in each fear conditioning chamber for foot shock delivery, using a different grid pattern for each context to differentiate floor texture between contexts. Next, place black plexiglass triangular inserts in context B to differentiate the internal layout of the two contexts. Wipe down the chamber walls and doors and spray the pans beneath the grid floors with a diluted cleaning solution.After the grid floor is cleaned, connect a shock generator and scrambler capable of delivering a 1 milliamp or lower amplitude shock to each grid floor for foot shock delivery. Finally, 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. On day one, set up context A with grid floors and visible light.Ensure the multimeter is attached to different bars to confirm the desired shock amplitude. Transport animals from the vivarium to the experimental room in their home cages placed on a cart, and place individually into the four fear conditioning chambers. For mouse experiments, use the shock generator and scramblers to deliver 10 one second, one milliamp foot shocks randomly presented over 60 minutes through the grid floors of the chambers containing trauma conditioned subjects.For rat experiments, use the shock generator and scramblers to deliver 15 one second, one milliamp foot shocks randomly presented over 90 minutes through the grid bars of the chambers containing trauma conditioned subjects. After 90 minutes for the rat, or 60 minutes for the mouse experiments, return all animals to their home cages and promptly return to the vivarium. On day two, set up context A and transport animals to the experimental room as done on day one.Place animals in context A for eight minutes, without shock delivery, and video record the behavior during the entire session. After eight minutes, return all animals to their home cages and promptly return to the vivarium. On day three, set up context B with a different set of grid floors from the ones used in context A and black triangular or white curved plexiglass inserts.Use infrared or near-infrared light if necessary to illuminate the chambers, and check the shock amplitude with the multimeter. Wipe down the chambers and spray the pans beneath the grid floors with the solution not used in context A.Next, transport animals from the vivarium to the experimental room in a black plastic tub, and place them individually into fear conditioning chambers. After a 180 second baseline period, deliver either a single one second, one milliamp foot shock for rats, or a single two second, one milliamp foot shock for mice, to all animals.Then, remove all animals 30 seconds after shock delivery and promptly return to the vivarium. On day four of the procedure, set up context B as done on day three. Transport animals from the vivarium to the experimental room in the same transport as done on day three.Place animals in context B for eight minutes without shock delivery, and video record freezing throughout the session. Finally, remove all animals after eight minutes and promptly return to the vivarium. Results indicate that 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.Both the trauma and no trauma animals showed minimal freezing levels that did not differ from each other. Further, the trauma animals showed lower shock reactivity compared to the no trauma controls, and trauma animals showed greater freezing compared to the no trauma controls, indicating that exposure to the traumatic stressor increased fear immediately following the mild stressor. Following this procedure, other methods like the elevated plus maze, open field, and light-dark box can be performed in order to answer additional questions like how trauma affects anxiety-like phenotypes.

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Behavior

11.8K Views.  University of California, Los Angeles. This method can help answer key questions in the behavioral neuroscience field, such as how to better understand underlying biological mechanisms of PTSD. The main advantage of this technique is it is robust, reliable, quantitative, and can be performed in multiple species. Begin by setting 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.Place each fear conditioning chamber inside a sound attenuating cubicle ...