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. 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>

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&#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...

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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 ...

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 ...

Compensatory Limb Use and Behavioral Assessment of Motor Skill Learning Following Sensorimotor Cortex Injury in a Mouse Model of Ischemic Stroke

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. The present protocol describes a skilled motor task for use in mouse models of stroke. The Pasta Matrix Reaching Task functions as a versatile and sensitive behavioral assay that permits experimenters to collect accurate outcome data and manipulate limb use to mimic human clinical phenomena including compensatory strategies (i.e., learned non-use) and focused rehabilitative training. When combined with neuroanatomical tools, this task also permits researchers to explore the mechanisms that support behavioral recovery of function (or lack thereof) following stroke. The task is both simple and affordable to set up and conduct, offering a variety of training and testing options for numerous research questions concerning functional outcome following injury. Though the task has been applied to mouse models of stroke, it may also be beneficial in studies of functional outcome in other upper extremity injury models.

Mouse models have become increasingly popular in studies of behavioral neuroscience. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. This protocol describes the Pasta Matrix Reaching Task, which is a skilled motor task for use in mouse models of stroke.

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models ...

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 ...

Disrupting Reconsolidation of Fear Memory in Humans by a Noradrenergic &#946;-Blocker

The basic design used in our human fear-conditioning studies on disrupting reconsolidation includes testing over different phases across three consecutive days. On day 1 - the fear acquisition phase, healthy participants are exposed to a series of picture presentations. One picture stimulus (CS1+) is repeatedly paired with an aversive electric stimulus (US), resulting in the acquisition of a fear association, whereas another picture stimulus (CS2-) is never followed by an US. On day 2 - the memory reactivation phase, the participants are re-exposed to the conditioned stimulus without the US (CS1-), which typically triggers a conditioned fear response. After the memory reactivation we administer an oral dose of 40 mg of propranolol HCl, a &#946;-adrenergic receptor antagonist that indirectly targets the protein synthesis required for reconsolidation by inhibiting the noradrenaline-stimulated CREB phosphorylation. On day 3 - the test phase, the participants are again exposed to the unreinforced conditioned stimuli (CS1- and CS2-) in order to measure the fear-reducing effect of the manipulation. This retention test is followed by an extinction procedure and the presentation of situational triggers to test for the return of fear. Potentiation of the eye blink startle reflex is measured as an index for conditioned fear responding. Declarative knowledge of the fear association is measured through online US expectancy ratings during each CS presentation. In contrast to extinction learning, disrupting reconsolidation targets the original fear memory thereby preventing the return of fear. Although the clinical applications are still in their infancy, disrupting reconsolidation of fear memory seems to be a promising new technique with the prospect to persistently dampen the expression of fear memory in patients suffering from anxiety disorders and other psychiatric disorders.

Disrupting reconsolidation is a promising approach to dampen the behavioral expression of fear memory in patients with anxiety disorders or posttraumatic stress disorder. In a series of human fear conditioning studies we showed that disrupting reconsolidation by the noradrenergic &#946;-blocker propranolol is very effective in erasing conditioned fear responding.

The basic design used in our human fear-conditioning studies on disrupting reconsolidation includes testing over different phases across three consecutive ...

Signal Attenuation as a Rat Model of Obsessive Compulsive Disorder

In the signal attenuation rat model of obsessive-compulsive disorder (OCD), lever-pressing for food is followed by the presentation of a compound stimulus which serves as a feedback cue. This feedback is later attenuated by repeated presentations of the stimulus without food (without the rat emitting the lever-press response). In the next stage, lever-pressing is assessed under extinction conditions (i.e., no food is delivered). At this stage rats display two types of lever-presses, those that are followed by an attempt to collect a reward, and those that are not. The latter are the measure of compulsive-like behavior in the model. A control procedure in which rats do not experience the attenuation of the feedback cue serves to distinguish between the effects of signal attenuation and of extinction. The signal attenuation model is a highly validated model of OCD and differentiates between compulsive-like behaviors and behaviors that are repetitive but not compulsive. In addition the measures collected during the procedure eliminate alternative explanations for differences between the groups being tested, and are quantitative, unbiased and unaffected by inter-experimenter variability. The major disadvantages of this model are the costly equipment, the fact that it requires some technical know-how and the fact that it is time-consuming compared to other models of OCD (11 days). The model may be used for detecting the anti- or pro-compulsive effects of pharmacological and non-pharmacological manipulations and for studying the neural substrate of compulsive behavior.

The goal of the protocol described in this paper is to induce compulsive-like behavior in rats for the study of obsessive-compulsive disorder (OCD). This behavior is precipitated by attenuating a signal indicating that a lever-press response was effective in producing food.

In the signal attenuation rat model of obsessive-compulsive disorder (OCD), lever-pressing for food is followed by the presentation of a compound stimulus ...

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

Quantifying Learning in Young Infants: Tracking Leg Actions During a Discovery-learning Task

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning process. Here a method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process. This discovery-learning task uses an infant activated mobile that rotates and plays music based on specified leg action of infants. Supine infants activate the mobile by moving their feet vertically across a virtual threshold. This paradigm is unique in that as infants independently discover that their leg actions activate the mobile, the infants&#8217; leg movements are tracked using a motion capture system allowing for the quantification of the learning process. Specifically, learning is quantified in terms of the duration of mobile activation, the position variance of the end effectors (feet) that activate the mobile, changes in hip-knee coordination patterns, and changes in hip and knee muscle torque. This information describes infant exploration and exploitation at the interplay of person and environmental constraints that support task-specific action. Subsequent research using this method can investigate how specific impairments of different populations of infants at risk for movement disorders influence the discovery-learning process for task-specific action.

A method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process.

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning ...

A Mouse Model of Subchronic and Mild Social Defeat Stress for Understanding Stress-induced Behavioral and Physiological Deficits

Stressful life events often increase the incidence of depression in humans. To study the mechanisms of depression, the development of animal models of depression is essential. Because there are several types of depression, various animal models are needed for a deeper understanding of the disorder. Previously, a mouse model of subchronic and mild social defeat stress (sCSDS) using a modified chronic social defeat stress (CSDS) paradigm was established. In the paradigm, to reduce physical injuries from aggressors, the duration of physical contact between the aggressor and a subordinate was reduced compared to in the original CSDS paradigm. sCSDS mice showed increased body weight gain, food intake, and water intake during the stress period, and their social behaviors were suppressed after the stress period. In terms of the face validity of the stress-induced overeating and overdrinking following the increased body weight gain, the sCSDS mice may show some features related to atypical depression in humans. Thus, a mouse model of sCSDS may be useful for studying the pathogenic mechanisms underlying depression. This protocol will help establish the sCSDS mouse model, especially for studying the mechanisms underlying stress-induced weight gain and polydipsia- and hyperphagia-like symptoms.

Here, methods for developing a mouse model of subchronic and mild social defeat stress are described and used to investigate the pathogenic features of depression including hyperphagia- and polydipsia-like symptoms following increased body weight.

Stressful life events often increase the incidence of depression in humans. To study the mechanisms of depression, the development of animal models of ...

Using Practice Testing, Public Speaking, and Source Monitoring to Examine the Influences of Learning Strategies and Stress on Episodic Memory

Prior research demonstrated that learning information via retrieval practice, which entails studying and taking practice tests, resulted in less memory impairment under stress than learning information via repeated studying. The present experiment combined three experimental procedures to further examine the memory mechanisms underlying the efficacy of retrieval practice in the context of stress. A list-discrimination task was implemented, in which participants learned two distinct wordlists. This was combined with a retrieval-practice manipulation, as half of the participants engaged in practice testing and half engaged in conventional studying during learning. A week later, participants underwent stress induction, using the Trier Social Stress Test. Before and after stress induction, participants completed tests of item and source memory (i.e., list discrimination). The combination of these three procedures yielded informative results: retrieval practice, in the context of stress, improved item memory but not source memory relative to conventional studying. Limitations and future directions for the use of this methodology are discussed.

The present experiment combined three experimental procedures &#8212; a retrieval-practice learning manipulation, a list-discrimination task, and a stress-induction technique &#8212; to examine the influences of different learning strategies and acute stress on multiple measures of episodic memory.

Prior research demonstrated that learning information via retrieval practice, which entails studying and taking practice tests, resulted in less memory ...