This work was funded by the U.S. Army Combat Capabilities Development Command (CCDC) Soldier Center. The views expressed in this article are solely those of the authors and do not reflect the official policies or positions of the U.S. Army, the Department of Defense, or any other department or agency of the U.S. government. The authors thank Abbie Gantner for her assistance with data collection.

This research was conducted in accordance with current American Psychological Association (APA) professional ethical standards and was approved by the Tufts University Social, Behavioral &#38; Educational Research Institutional Review Board.&#160;
NOTE: The experiment reported in the present manuscript was previously published elsewhere13.
1. Participant Recruitment, Screening, and Scheduling
<ol>
	<li>Conduct a power analysis to determine the sample size needed to achieve the desired power and detect the expected effect size. The free software package G*Power14 is useful for this step.
	<ol>
		<li>After opening G*Power, select F tests from the Test Family dropdown menu.</li>
		<li>Select ANOVA: Repeated measures, within-between interaction from the Statistical test dropdown menu.</li>
		<li>Select the A priori option from the Type of power analysis dropdown menu.</li>
		<li>Enter the predicted effect size in the Effect size f box.</li>
		<li>Enter the desired alpha value in the &#945; err prob box.</li>
		<li>Enter the desired power value in the Power (1-&#946; err prob) box.</li>
		<li>Enter 2 in the Number of groups box.</li>
		<li>Enter 2 in the Number of measurements box.</li>
		<li>Enter 0.7 in the Corr among rep measures box13.</li>
		<li>Click Calculate on the bottom right of the window. The total sample size needed for the experiment will be displayed in the Total sample size box.</li>
		<li>Recruit 10-20 participants beyond the recommended sample size to account for participant error or dropout.</li>
	</ol>
	</li>
	<li>Recruit participants in the desired age range (e.g., 18-24 years old) who are native English speakers, are not color-blind, and have normal or corrected-to-normal vision. If interested in a representative sample, recruit both male and female participants. 
	NOTE: Women sometimes demonstrate a blunted cortisol response to psychological stress, particularly when in the follicular phase of their menstrual cycle or when taking oral contraceptives15. Consider measuring/controlling for female use of contraceptives and menstrual cycle phase (this was not done in the present study).</li>
	<li>When scheduling, ensure that participants can attend both testing sessions, which are approximately 1 h each and occur one week apart.</li>
	<li>Schedule participants in groups of two to facilitate peer evaluation during stress induction.</li>
	<li>To ensure that cortisol samples are not biased by the use of stimulants, ask participants to refrain from stimulant use (e.g., caffeine, nicotine) for 6 h prior to each experimental session. To avoid contamination of saliva samples. Also ask participants to refrain from eating or drinking (besides water) for 1 h prior to each session.</li>
	<li>Randomly assign participants to either learning group: study-practice or retrieval-practice.</li>
	<li>Schedule all participants during roughly the same time of day (e.g., all morning testing or all evening testing) to control for diurnal variability in cortisol16. Schedule each participant&#8217;s two experimental sessions for the same time (one week apart).</li>
</ol>
2. Construction of Stimuli and Memory Tests
<ol>
	<li>Encoding stimuli
	<ol>
		<li>In the interest of creating two dissociable learning events, create two distinct sets of materials for participants to encode. 
		NOTE: This protocol involved two 60-item wordlists, one of which was labelled &#34;The Red List&#34;&#160;and was visually presented in red font, the other of which was labelled &#34;The Blue List&#34;&#160;and was visually presented in blue font. The wordlists used in the present protocol are available in Supplementary File 1.</li>
		<li>For the purposes of stimulus neutrality, ensure that each word meets the following criteria: (1) non-proper noun, (2) not a homograph, (3) four to eight letters long, and (4) concreteness rating of at least 4 on a scale from 1-7 (7 = most concrete). Compare the words to valence and arousal norms17 to ensure that that they have valence ratings between 4.00 and 5.99 on a 1-9 scale (i.e., neutral valence) and arousal ratings lower than 4.00 on a 1-9 scale (i.e., not negatively arousing). Equate word frequencies across the lists by comparing them to an established word database18.</li>
		<li>Use stimulus-presentation software to program each of the wordlists such that words are individually and randomly presented at a rate of 2 s per word. 
		NOTE: This protocol used E-Prime version 2.119 for stimulus presentation, but common software can be used to this effect. The E-Prime scripts used for stimulus presentation in the present experiment are available in Supplementary File 2.</li>
	</ol>
	</li>
	<li>Retrieval stimuli
	<ol>
		<li>To create plausible foil items for the recognition memory test, create a third 60-item wordlist. Adhere to the criteria outlined in step 2.1.2. The list of foil words used in the present protocol is available in Supplementary File 1.</li>
	</ol>
	</li>
	<li>Memory tests
	<ol>
		<li>For the purposes of assessing memory performance both pre- and post-stress, create two functionally-identical memory tests. Construct two 90-item tests. 
		NOTE: The present study used recognition tests, in which participants are presented with studied items and foil items and must indicate whether each item occurred on the lists they studied.</li>
		<li>Tests consist of 30 items from the red list, 30 items from the blue list, and 30 items from the foil list. Counterbalance or randomize which list items are presented on the two recognition tests. Also randomize the order in which the items are presented. 
		NOTE: Each item is accompanied by a list-discrimination question and an assessment of confidence. The list-discrimination question asks participants to indicate whether the item came from the red list, the blue list, or neither list. The confidence question asks participants whether they experienced high or low confidence in their list-discrimination judgment.</li>
	</ol>
	</li>
</ol>
3. Stress-induction Protocol
<ol>
	<li>Use the Trier Social Stress Test for Groups (TSST-G)20 to induce mild, acute psychological stress. 
	NOTE: The present protocol adapted the TSST-G to test participants in groups of two instead of four. Psychosocial stress induction of this nature, which involves giving a speech and solving math problems while being evaluated, is preferable to other methods of stress induction because of its ecological validity and ability to elicit a physiological stress response21,22.</li>
	<li>To induce stress, do the following in this order:
	<ol>
		<li>Speech preparation
		<ol>
			<li>Have participants sit at separate desks. Give both participants a sheet of blank paper and a pen or pencil. Read them the following instructions: 
			&#34;You will now have two minutes to prepare a speech in which you are applying for a job as a Teaching Assistant* in any course of your choice. Please be prepared to discuss what skills and experiences you have that make you a qualified candidate for the job&#34; 
			NOTE: *If working with non-student populations, change &#34;Teaching Assistant&#34;&#160;to a population-relevant position.</li>
			<li>Use a stopwatch or clock to time the 2 min period. Set up a video camera on a tripod while participants are preparing their speeches. Take participants&#39;&#160;notes away when two minutes have passed.</li>
		</ol>
		</li>
		<li>Speech delivery
		<ol>
			<li>Read participants the following instructions: 
			&#34;You have each been assigned a number on your desk. I will now call on you by number to stand and give your speech. When I call on you, please stand in the center of the room where you can be seen by the camera. You will be video recorded for the purpose of coding your non-verbal behavior at a later time.&#34;</li>
			<li>Call on participants one at a time to stand up and deliver their speeches. Use a stopwatch or clock to ensure that each speech lasts for 2 min. If a participant finishes the speech early, tell them that they still have time left and they must continue.</li>
		</ol>
		</li>
		<li>Oral subtraction
		<ol>
			<li>Read participants the following instructions: 
			&#34;I will now call on you at random to solve simple math subtraction problems. When I call on you, please stand and solve the problem aloud. If you get the problem wrong, I will ask you to try again until you get it correct. As a reminder, you will be videotaped during this part of the experiment. Person X, please stand and subtract...&#34; 
			NOTE: Math problems involve subtracting a number in the teens (e.g., 13, 14, 15) from a 4-digit number (e.g., 4,563).</li>
			<li>Call on participants randomly and unpredictably (e.g., sometimes 2-3 times in a row) to solve math problems for 6 min. That is, participants solve math problems multiple times each for a period of 6 min. When participants give an incorrect answer, tell them they are incorrect and ask them to try again. Repeat the question if necessary.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Saliva Sample Collection and Supplies
NOTE: Saliva collection is only necessary during experimental session 2, in which stress is induced
<ol>
	<li>Ensure that participants have adhered to the instructions outlined in step 1.5. If any of these criteria have been violated, make note of this and examine the participant&#39;s data as a potential outlier before conducting any statistical analyses.</li>
	<li>When participants first arrive for this session, have them rinse their mouth with water to clear it of any sample contaminants.</li>
	<li>For the collection of saliva samples, use the passive drool method in which participants pass saliva through a straw into a collection tube. For sample collection, use smoothie-sized straws, cut into 2 inch pieces, and 2 mL cryovials. Ensure that all cryovials are labelled with necessary identifying information (e.g., participant number, sample number, date).
	<ol>
		<li>For each sample, have the participant place the 2 inch straw in the cryovial and drool into the straw until they have provided 2 mL of saliva. When they have finished providing a sample, have them discard the straw in a wastebasket and screw the cap on the cryovial. Offer participants gloves and sanitizing wipes before they provide each saliva sample.</li>
	</ol>
	</li>
	<li>Collect three saliva samples: (1) a baseline sample after participants arrive for session 2, (2) a sample immediately after stress induction is complete (~12 min after the onset of stress), and (3) a sample 25-30 min after the onset of stress induction, when cortisol reaches its peak post-stress levels in saliva12.</li>
	<li>Store the saliva samples in a freezer (0 &#176;F) until analyzed.</li>
</ol>
5. Subjective Stress Measure
<ol>
	<li>Choose a measurement of self-reported state anxiety/stress to administer at various times during the experiment. 
	NOTE: The present protocol used the State-Trait Inventory for Cognitive and Somatic Anxiety (STICSA)23. The STICSA is a state measure of the cognitive and somatic sensations associated with anxiety. Statements are rated on a four-point Likert-type scale, and responses are added for a total score that ranges from 0-80 (higher scores are indicative of greater anxiety).</li>
	<li>Before administering each iteration of the STICSA, inform participants that it is a measure of anxiety and they should respond based on how they are feeling in the moment.</li>
</ol>
6. Encoding Procedure (Experimental Session 1)
<ol>
	<li>Instruct participants to read the informed consent form and, if they choose to participate, sign and return it to the experimenter.</li>
	<li>Instruct participants that that they will be presented with a series of words that they must try to remember for a later test.</li>
	<li>Present participants with either the Red List or the Blue List using the stimulus-presentation software and rate of presentation outlined in section 2.1.</li>
	<li>The next step depends on group assignment.
	<ol>
		<li>For participants in the study-practice group, present the same list two more times at the same rate of presentation. To clear working memory between each study event, have participants complete simple math problems for 30 s (e.g., 12 x 4). Either have participants complete this task with pencil and paper or embed the task into the computer program used for stimulus presentation. 
		NOTE: The scripts included in Supplementary File 2 have all encoding tasks embedded within them.</li>
		<li>For participants in the retrieval-practice group, give them two time-matched (i.e., 2 min) free-recall tests. Prior to the two tests, instruct participants that they should recall as many words as possible from the preceding list, in any order. During free recall, either have participants type their responses using the stimulus-presentation software program or have them write their responses on a sheet of paper. To clear working memory, have participants complete simple math problems between initial presentation of the wordlist and the first test, as well as between the two free-recall tests (see step 6.4.1).</li>
	</ol>
	</li>
	<li>Present participants with a 30 min clip from an emotionally-neutral movie or television show. 
	NOTE: The present protocol used the BBC television series Planet Earth. This 30 min delay helps establish a temporal distinction between learning the Red List and learning the Blue List.</li>
	<li>Repeat steps 6.3 and 6.4 for the list that was not presented prior to the 30 min break. 
	NOTE: Steps 6.3 through 6.6 are depicted in Figure 1.</li>
	<li>Have participants complete a first iteration of the STICSA to determine whether study practice and retrieval practice differentially influenced anxiety levels.</li>
</ol>
7. Retrieval Procedure (Experimental Session 2)
<ol>
	<li>Follow steps 1.5 and 4.2 to ensure proper saliva sample collection.</li>
	<li>Instruct participants to fill out a second STICSA as a pre-stress measure of subjective anxiety.</li>
	<li>Instruct participants to provide the first saliva sample.</li>
	<li>Administer one of the two tests constructed in section 2.3. 
	NOTE: The tests are depicted in Figure 2.</li>
	<li>Complete the stress-induction procedure as specified in section 3.2.</li>
	<li>Instruct participants to complete the third STICSA as a post-stress measure of subjective anxiety.</li>
	<li>Instruct participants to provide the second saliva sample.</li>
	<li>Give participants a 10 min break. In the present protocol, participants watched part of an episode of The Office during this break.</li>
	<li>Instruct participants to provide the third saliva sample.</li>
	<li>Administer the second test constructed in section 2.3 (depicted in Figure 2).</li>
	<li>Debrief participants about the purpose of the experiment and excuse them.</li>
</ol>
8. Computing Dependent Measures
<ol>
	<li>After the conclusion of data collection, compute the dependent measures as follows:
	<ol>
		<li>Calculate hit proportions. For each participant, divide the number of studied items that participants correctly recognized by the total number of studied items that were presented on the recognition test.</li>
		<li>Calculate false-alarm proportions. For each participant, divide the number of non-presented foil words that participants falsely recognized by the total number of foils that were presented on the recognition test.</li>
		<li>Calculate source-memory scores. For each participant, divide the total number of hits that participants attributed to the correct source by their total number of hits.</li>
		<li>For average confidence, code the high- and low-confidence judgments as binary, with 1 representing high confidence and 0 representing low confidence. For each participant, calculate their proportion of high-confidence judgments.</li>
		<li>Calculate gamma correlations. For each participant, correlate their accuracy on each list-discrimination question with each accompanying confidence rating.</li>
		<li>Calculate delta cortisol. For each participant, subtract their baseline cortisol concentration from the cortisol concentration of the sample taken 25 min post-stress.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Shields, G. S., Sazma, M. A., McCullough, A. M., Yonelinas, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+acute+stress+on+episodic+memory:+a+meta-analysis+and+integrative+review.">The effects of acute stress on episodic memory: a meta-analysis and integrative review.</a> Psychological Bulletin. 143 (6), 636-675 (2017).</li><li>Buchanan, T. W., Tranel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+and+emotional+memory+retrieval:+effects+of+sex+and+cortisol+response.">Stress and emotional memory retrieval: effects of sex and cortisol response.</a> Neurobiology of Learning and Memory. 89 (2), 134-141 (2008).</li><li>Domes, G., Heinrichs, M., Rimmele, U., Reichwald, U., Hautzinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+stress+impairs+recognition+for+positive+words--association+with+stress-induced+cortisol+secretion.">Acute stress impairs recognition for positive words--association with stress-induced cortisol secretion.</a> Stress: The International Journal on the Biology of Stress. 7 (3), 173-181 (2004).</li><li>Hidalgo, V., 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=Acute+stress+affects+free+recall+and+recognition+of+pictures+differently+depending+on+age+and+sex.">Acute stress affects free recall and recognition of pictures differently depending on age and sex.</a> Behavioral Brain Research. 292, 393-402 (2015).</li><li>Kuhlmann, S., Piel, M., Wolf, O. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+memory+retrieval+after+psychosocial+stress+in+healthy+young+men.">Impaired memory retrieval after psychosocial stress in healthy young men.</a> Journal of Neuroscience. 25 (11), 2977-2982 (2005).</li><li>Merz, C. J., Wolf, O. T., Hennig, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+impairs+retrieval+of+socially+relevant+information.">Stress impairs retrieval of socially relevant information.</a> Behavioral Neuroscience. 124 (2), 288-293 (2010).</li><li>Lovallo, W. R., Robinson, J. L., Glahn, D. C., Fox, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+effects+of+hydrocortisone+on+the+human+brain:+An+fMRI+study.">Acute effects of hydrocortisone on the human brain: An fMRI study.</a> Psychoneuroendocrinology. 35 (1), 15-20 (2010).</li><li>Reul, J. M., de Kloet, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+receptor+systems+for+corticosterone+in+rat+brain:+Microdistribution+and+differential+occupation.">Two receptor systems for corticosterone in rat brain: Microdistribution and differential occupation.</a> Endocrinology. 117 (6), 2505-2511 (1985).</li><li>Gagnon, S. A., Waskom, M. L., Brown, T. I., Wagner, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=stress+impairs+episodic+retrieval+by+disrupting+hippocampal+and+cortical+mechanisms+of+remembering.">stress impairs episodic retrieval by disrupting hippocampal and cortical mechanisms of remembering.</a> Cerebral Cortex. 162, 1-18 (2018).</li><li>Smith, A. M., Floerke, V. A., Thomas, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrieval+practice+protects+memory+against+acute+stress.">Retrieval practice protects memory against acute stress.</a> Science. 354 (6315), 1046-1048 (2016).</li><li>Karpicke, J. D., Lehman, M., Aue, W. R., Ross, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrieval-based+learning:+An+episodic+context+account.">Retrieval-based learning: An episodic context account.</a> The Psychology of Learning and Motivation. , 237-284 (2014).</li><li>Kirschbaum, C., Pirke, K., Hellhammer, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"trier+social+stress+test":+A+tool+for+investigating+psychobiological+stress+responses+in+a+laboratory+setting.">The "trier social stress test": A tool for investigating psychobiological stress responses in a laboratory setting.</a> Neuropsychobiology. 28 (1-2), 76-81 (1993).</li><li>Smith, A. M., Race, E., Davis, F. C., Thomas, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrieval+practice+improves+item+memory+but+not+source+memory+in+the+context+of+stress.">Retrieval practice improves item memory but not source memory in the context of stress.</a> Brain and Cognition. , (2018).</li><li>Faul, F., Erdfelder, E., Lang, A. G., Buchner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G*Power+3:+A+flexible+statistical+power+analysis+program+for+the+social,+behavioral,+and+biomedical+sciences.">G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences.</a> Behavioral Research Methods. 39, 175-191 (2007).</li><li>Kajantie, E., Phillips, D. I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+sex+and+hormonal+status+on+the+physiological+response+to+acute+psychosocial+stress.">The effects of sex and hormonal status on the physiological response to acute psychosocial stress.</a> Psychoneuroendocrinology. 31 (2), 151-178 (2006).</li><li>Weitzman, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty-four+hour+pattern+of+the+episodic+secretion+of+cortisol+in+normal+subjects.">Twenty-four hour pattern of the episodic secretion of cortisol in normal subjects.</a> The Journal of Clinical Endocrinology &amp; Metabolism. 33 (1), 14-22 (1971).</li><li>Warriner, A. B., Kuperman, V., Brysbaert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norms+of+valence,+arousal,+and+dominance+for+13,915+English+lemmas.">Norms of valence, arousal, and dominance for 13,915 English lemmas.</a> Behavior Research Methods. 45, 1191-1207 (2013).</li><li>Brysbaert, M., New, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+beyond+Kucera+and+Francis:+A+critical+evaluation+of+current+word+frequency+norms+and+the+introduction+of+a+new+and+improved+word+frequency+measure+for+American+English.">Moving beyond Kucera and Francis: A critical evaluation of current word frequency norms and the introduction of a new and improved word frequency measure for American English.</a> Behavior Research Methods. 41 (4), 977-990 (2009).</li><li>Schneider, W., Eschman, A., Zuccolotto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-Prime+user's+guide.">E-Prime user's guide.</a> Psychology Software Tools. , (2001).</li><li>von Dawans, B., Kirschbaum, C., Heinrichs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Trier+Social+Stress+Test+for+Groups+(TSST-G):+A+new+research+tool+for+controlled+simultaneous+social+stress+exposure+in+a+group+format.">The Trier Social Stress Test for Groups (TSST-G): A new research tool for controlled simultaneous social stress exposure in a group format.</a> Psychoneuroendocrinology. 36 (4), 514-522 (2011).</li><li>Dickerson, S. S., Kemeny, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+stressors+and+cortisol+responses:+A+theoretical+integration+and+synthesis+of+laboratory+research.">Acute stressors and cortisol responses: A theoretical integration and synthesis of laboratory research.</a> Psychological Bulletin. 130 (3), 355-391 (2004).</li><li>Skoluda, 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=Intra-individual+psychological+and+physiological+responses+to+acute+laboratory+stressors+of+different+intensity.">Intra-individual psychological and physiological responses to acute laboratory stressors of different intensity.</a> Psychoneuroendocrinology. 51, 227-236 (2015).</li><li>Gr&#246;s, D. F., Antony, M. M., Simms, L. J., McCabe, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychometric+properties+of+the+state-trait+inventory+for+cognitive+and+somatic+anxiety+(STICSA):+Comparison+to+the+state-trait+anxiety+inventory+(STAI).">Psychometric properties of the state-trait inventory for cognitive and somatic anxiety (STICSA): Comparison to the state-trait anxiety inventory (STAI).</a> Psychological Assessment. 19 (4), 369-381 (2007).</li><li>Kang, S. H. K., McDermott, K. B., Roediger, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test+format+and+corrective+feedback+modify+the+effect+of+testing+on+long-term+retention.">Test format and corrective feedback modify the effect of testing on long-term retention.</a> European Journal of Cognitive Psychology. 19 (4-5), 528-558 (2007).</li><li>Tulving, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+presentation+and+recall+of+material+in+free-recall+learning.">The effects of presentation and recall of material in free-recall learning.</a> Journal of Verbal Learning and Verbal Behavior. 6, 175-184 (1967).</li><li>Brewer, G. A., Marsh, R. L., Meeks, J. T., Clark-Foos, A., Hicks, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+free+recall+testing+on+subsequent+source+memory.">The effects of free recall testing on subsequent source memory.</a> Memory. 18 (4), 385-393 (2010).</li><li>Chan, J. C. K., McDermott, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+testing+effect+in+recognition+memory:+A+dual+process+account.">The testing effect in recognition memory: A dual process account.</a> Journal of Experimental Psychology: Learning, Memory, and Cognition. 33 (2), 431-437 (2007).</li><li>Smith, A. M., Davis, F. C., Thomas, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Criterial+learning+is+not+enough:+Retrieval+practice+is+necessary+for+improving+post-stress+memory+accessibility.">Criterial learning is not enough: Retrieval practice is necessary for improving post-stress memory accessibility.</a> Behavioral Neuroscience. 132 (3), 161-170 (2018).</li><li>Smith, A. M., Hughes, G. H., Davis, F. C., Thomas, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+stress+enhances+general-knowledge+semantic+memory.">Acute stress enhances general-knowledge semantic memory.</a> Hormones and Behavior. 109, 38-43 (2019).</li><li>Domes, G., Heinrichs, M., Rimmele, U., Reichwald, U., Hautzinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+stress+impairs+recognition+for+positive+words--association+with+stress-induced+cortisol+secretion.">Acute stress impairs recognition for positive words--association with stress-induced cortisol secretion.</a> Stress: The International Journal on the Biology of Stress. 7 (3), 173-181 (2004).</li><li>Pulopulos, M. M., Almela, M., Hidalgo, V., Villada, C., Puig-Perez, S., Salvador, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+stress+does+not+impair+long-term+memory+retrieval+in+older+people.">Acute stress does not impair long-term memory retrieval in older people.</a> Neurobiology of Learning and Memory. 104, 16-24 (2013).</li></ol>

The authors have nothing to disclose.

Episodic memories are associated with contextual information. To gain a deeper understanding of how psychological stress and different learning strategies influence episodic memory, it is important to consider how these variables influence the contextual elements of memory. The context associated with a memory (e.g., where the memory was acquired, when it was acquired) could be examined in infinitely-many ways. The present protocol took a step forward in examining memory for context with the use of a list-discrimination task, which assesses the source of a given memory. The novel combination of a list-discrimination task with stress induction and a retrieval-practice encoding manipulation allowed for a test of the memory mechanisms underlying the efficacy of retrieval practice for buffering memory against acute stress10.
Future researchers may consider modifying the retrieval-practice manipulation employed in the present protocol. As stated in the Results, participants in the retrieval-practice group only recalled about 25% of wordlist items during their free-recall attempts in session 1. This low level of performance is not optimal either experimentally or practically, although the hit-proportion restriction used in the present protocol did allow for meaningful examination of the efficacy of retrieval practice. For consistency with previous literature10, the present protocol required that the retrieval-practice group simply study each wordlist once and then make free-recall attempts without subsequent feedback or re-exposure to the stimuli. However, research suggests that retrieval practice results in more robust learning when participants are given feedback about their performance during the retrieval-practice attempts24 and when participants are re-exposed to the stimuli between each retrieval-practice attempt25. Thus, a more effective retrieval-practice manipulation may involve multiple cycles of studying, free-recall testing, and feedback regarding correctness.
The list-discrimination procedure employed in the present experiment was only somewhat effective, given that some participants demonstrated chance-level performance. This task was borrowed from previous research, in which source memory was tested within minutes of the wordlist encoding procedure26,27. The one-week delay between encoding and memory testing that was implemented in the present protocol may have contributed to the observed chance-level list-discrimination performance. Thus, to improve list-discrimination performance so that any effects of stress and retrieval practice may become more apparent, future researchers may consider shortening the retention interval between encoding and testing. However, the one-week interval used in the present protocol may be desirable because it mimics more realistic circumstances (e.g., learning information today and recalling it a week from now). To maintain this delay but improve participants&#39;&#160;ability to discriminate between items learned from the two wordlists, researchers may consider making the two encoding episodes (i.e., learning the two wordlists) more distinct. As examples, encoding of the two wordlists could be separated by a longer interval of time or the wordlists could be encoded in different physical locations.
In addition to the limitations of the retrieval-practice and list-discrimination protocols, the stress-induction procedure used in the present experiment demonstrated limited efficacy. Participants showed only a moderate increase in cortisol across the three measurements. Because this exact method of stress-induction has successfully induced stronger stress responses in previous experiments28,29, the present results are likely due to sample differences rather than an ineffective paradigm. Women, particularly those taking oral contraceptives, often demonstrate a blunted cortisol response to acute stressors15. The present study recruited a largely-female sample (73% female), which may have contributed to lower post-stress cortisol at the group level. There are several options for controlling for this issue. Future researchers may choose to recruit a male-only sample30, include sex as an independent variable in statistical analyses4, or include contraceptive use and menstrual-cycle phase as variables in analyses on cortisol1. However, these options require additional considerations. The first limits the generalizability of findings, and the second and third require larger sample sizes to account for the addition of variables to the statistical model.
Some additional methodological changes should be considered. First, to better map the post-stress increase in cortisol and subsequent recovery period, researchers should collect more saliva samples. For example, some researchers opt to collect samples every 5-10 min after the onset of stress for up to one hour after stress induction31. Second, researchers may consider manipulating stress between-subjects. In the present repeated-measures design, issues such as participant fatigue could confound the effects of stress that were observed. A between-subjects design with a non-stressed control group would eradicate these potential issues. Additionally, the act of retrieving items on the pre-stress test may have imparted retrieval-practice benefits on the post-stress test, effectively reducing the benefits of the retrieval-practice encoding manipulation. A between-subjects manipulation of stress, featuring only one memory test (post-stress), would eliminate this potential issue. Third, researchers should consider the emotional impact of the distractor task implemented between stress induction and memory testing during session 2. Having participants watch a sitcom (i.e., The Office) may induce a positive mood. Including a post-sitcom measure of participants&#39;&#160;mood states would provide a more precise understanding of how mood and physiological stress influence subsequent memory performance. As a final note, an additional baseline measure of state anxiety should be added to the beginning of session 1 in the present protocol. To examine if study practice and retrieval practice differentially impact anxiety levels during session-1 encoding, this initial measure is necessary for comparison to the measure that is taken at the end of session 1.
The present protocol employed a novel combination of three experimental procedures &#8212; a retrieval-practice manipulation, a list-discrimination task, and the TSST stress-induction technique &#8212; to examine one potential memory mechanism underlying the effectiveness of retrieval practice at creating stress-resistant memories. The results of this methodology showed that, in the context of stress, retrieval practice improved memory for the items that were learned but did not improve memory for the source of the items. These findings speak to the efficacy of combining these procedures to investigate questions related to retrieval practice, the contextual elements of episodic memory, and/or acute psychological stress. Future research examining questions related to one or more of these topics should consider using these techniques, but with attention given to the modifications outlined above.

Instances of acute psychological stress generally impair memory retrieval1. For instance, performing a high-pressure public-speaking task reduces the amount of information that individuals can subsequently remember2,3,4,5,6. This common finding is largely attributed to the neural influence of the human stress hormone cortisol. When cortisol levels are heightened after stress, cortisol binds to glucocorticoid receptors in the hippocampus7,8, impairing recollection of previously-learned information9.
In recent research, the detrimental effects of stress on memory retrieval were eliminated when participants studied stimuli using the highly-effective learning strategy retrieval practice10. Participants either learned a wordlist via repeated studying, or via studying followed by several attempts at freely recalling the words (i.e., retrieval practice). A day later, when memory was tested after stress induction, those who learned via repeated studying remembered fewer words than their non-stressed counterparts, whereas those who learned via repeated testing showed no memory impairment.
To better understand why retrieval practice so effectively buffered memory against the deleterious effects of acute stress, it is helpful to take a closer look at the memory processes being affected. The prior study simply examined item memory by having participants recall the words that they had learned10. In the present study, source memory was more carefully examined to determine whether retrieval practice was also supporting memory for the context in which each item was learned. This approach was informed by research showing that retrieval practice increases memory for contextual information associated with a given learning episode (e.g., when each item was learned, where each item was learned)11.
To determine how retrieval practice interacts with acute stress to influence both item and source memory, it was necessary to combine three experimental procedures. First, a standard retrieval-practice manipulation was implemented in which participants either studied stimuli several times or engaged in studying followed by free recall. Second, a list-discrimination task was used to separately examine item and source memory. This involved having participants learn stimuli from two temporally-segregated and color-coded lists that could be differentiated on a later memory test. Third, a commonly-used method of psychological stress induction was used, in which participants must give a short speech and solve math problems while being judged12.

<table border="0" cellpadding="0" cellspacing="0" style="width:867px;" width="866">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Blank paper</td>
			<td style="width:119px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:327px;">Paper is given to participants for speech preparation during stress induction</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:303px;">Clipboard equipped with blank sheets of paper and a list of 50 math subtraction tasks with answers (e.g., 4782 - 17).</td>
			<td style="width:119px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:327px;">The experimenter holds the clipboard during stress induction. (S)he takes notes on the blank paper during participants&#39; speeches, and refers to the math problems for the math-subtraction portion of stress induction.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:303px;">Computers (two)</td>
			<td style="width:119px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:327px;">Necessary for presenting stimuli, administering memory tests, and recording participants&#39; responses.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Crovial storage box (recommended)</td>
			<td style="width:119px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:327px;">Helpful for storing saliva samples until the time of analysis.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:303px;">Cryovials (2 mL)</td>
			<td style="width:119px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:327px;">Needed for collection of saliva samples for cortisol analysis. The present paradigm required three cryovials per participant.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Cubicle desks with chairs (two of each)</td>
			<td style="width:119px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:327px;">Cubicles are helpful for limiting distractions during encoding and memory testing.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:303px;">Freezer</td>
			<td style="width:119px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:327px;">Needed for storing saliva samples until the time of analysis. Freezer must maintain a temperature of approximately 0 degrees fahrenheit.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Smoothie-sized straws</td>
			<td style="width:119px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:327px;">Helpful for passing saliva from the mouth into each cryovial.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:303px;">Stimulus presentation software (recommended)</td>
			<td style="width:119px;">E-Prime 3.0 (recommended)</td>
			<td style="width:119px;"></td>
			<td style="width:327px;">Helpful for precision in stimulus presentation (e.g., 2 s presentation rate for words during experimental session 1) and video presentation (e.g., Planet Earth during session 1). Also helpful for administration of recognition tests and recording participant responses (session 2).</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:303px;">The State Trait Inventory for Cognitive and Somatic Anxiety</td>
			<td style="width:119px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:327px;">Necessary for assessing subjective stress. Borrowed from Gr&#246;s, Antony, Simms, and McCabe (2007).</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:303px;">Video camera with tripod</td>
			<td style="width:119px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:327px;">Necessary for stress-induction procedure. Camera needs to appear to record participants, but does not need to be capable of recording.</td>
		</tr>
	</tbody>
</table>

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.

Efficacy of the retrieval-practice manipulation 
Note that the following analyses were previously published by Smith et al.13. During encoding in experimental session 1, free recall of the words from each wordlist was relatively low for individuals in the retrieval-practice group. These individuals recalled, on average, 14 of 60 items and 13 of 60 items during their recall attempts for the first list. They recalled, on average, 16 of 60 items and 15 of 60 items during their recall attempts for the second list.
Of note is the fact that retrieval practice is only beneficial as a learning technique if the retrieval-practice attempts that one makes are successful. That is, an individual must successfully retrieve the studied information during their retrieval-practice attempts to experience any subsequent memory benefits. For example, an individual who recalls only 5 of 60 items during their retrieval-practice attempts would not show exceptional memory performance on a later test, while an individual who recalls 55 of the items likely would. Therefore, hit proportions on the final recognition tests for individuals in the retrieval-practice group were restricted to items that they accurately recalled at least once during session 1, in order to meaningfully examine the benefits of retrieval practice.
Using this restriction, the retrieval-practice group demonstrated higher hit proportions on the session 2 recognition tests than those in the study-practice group, as expected [F(1, 60) = 80.34, p &#60; 0.001, &#951;p2 = 0.57]. Individuals in the retrieval-practice group demonstrated average hit rates of 0.91 (standard error of the mean [SEM] = 0.02) and 0.91 (SEM = 0.01) on the pre- and post-stress tests (respectively), whereas those in the SP group had average hit proportions of 0.66 (SEM = 0.02) and 0.66 (SEM = 0.03).
Efficacy of the list-discrimination task 
To determine whether performance on the list-discrimination task was due to participants&#8217; ability to discriminate between items learned on the two lists or was simply due to guessing, source memory scores were compared to chance levels of performance (i.e., 50% accuracy). Participants in the study-practice group exhibited above-chance levels of discrimination on both the pre- and post-stress recognition tests [pre-stress test: t(29) = 3.14, p = 0.004; post-stress test: t(29) = 2.78, p = 0.009]13. However, those in the retrieval-practice group demonstrated chance levels of performance on both tests [pre-stress test: t(31) = 0.76, p = 0.452; post-stress test: t(31) = 1.50, p = 0.144]13. Note that participants had accurately recalled these items at least once during their session-1 free-recall attempts, but still demonstrated chance levels of performance.
Efficacy of the stress-induction procedure 
As shown in Figure 3, the modified version of the TSST-G used in the present protocol effectively induced both psychological stress and physiological stress. Participants demonstrated post-stress increases in STICSA scores [F(1, 60) = 25.93, p &#60; 0.001, &#951;p2 = 0.30] and cortisol levels [F(2, 116) = 3.75, p = 0.026, &#951;p2 = 0.06]. Across the three measures of cortisol, participants demonstrated marked increases from baseline to 25 min post-stress [t(59) = 1.97, p = 0.027, Cohen&#8217;s d = 0.25] and from 12 min post-stress to 25 min post-stress [t(59) = 2.16, p = 0.018, Cohen&#8217;s d = 0.28]. Cortisol levels did not significantly increase from baseline to 12 min post-stress [t(59) = 1.24, p = 0.110].
Putting it all together: the influence of stress and retrieval practice on item and source memory 
Across the measures of item memory, the combination of stress and retrieval practice generally produced the best performance on the session-2 recognition tests. The retrieval-practice group demonstrated a positive relationship between delta cortisol and hit proportions [r(31) = 0.41, p = 0.023], whereas the study-practice group did not [r(27) = -0.17, p = 0.384]. As depicted in Figure 4, the retrieval-practice group also demonstrated the lowest false-alarm proportions, but only on the post-stress test [F(1, 60) = 4.10, p = 0.047, &#951;p2 = 0.06]. Last, whereas gamma correlations were generally at chance levels for list-discrimination, the post-stress gamma value for the retrieval-practice group was the only value to exceed chance-level performance [t(31) = 3.03, p = 0.005, d = 0.54]. This reflects participants&#39;&#160;awareness of which items they correctly and incorrectly assigned to red and blue lists.
In contrast, source memory was unaffected by stress and, as noted above, individuals in the retrieval-practice group demonstrated chance-level performance (Figure 4). Despite correctly remembering 91% of the items that they correctly recalled during free-recall in session 1, individuals in the retrieval-practice group could not accurately remember whether these items came from the red or the blue list.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60026/60026fig1.jpg" /> 
Figure 1: Graphic simulation of the encoding procedure during session 1. The stimuli depicted are representative of the two 60-item lists of stimuli. <a href="https://www.jove.com/files/ftp_upload/60026/60026fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60026/60026fig2.jpg" /> 
Figure 2: Graphic simulation of the recognition test and accompanying list-discrimination questions and confidence judgments. Each recognition question is presented individually, followed by a prompt to indicate which list the item came from and how confident the participant is in that judgment. Ninety items are presented in this manner on each recognition test (see section 2.3). <a href="https://www.jove.com/files/ftp_upload/60026/60026fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60026/60026fig3.jpg" /> 
Figure 3: Evidence of the efficacy of the TSST stress-induction procedure. Stress increased subjective anxiety, as measured by the STICSA (A), and also increased salivary cortisol levels relative to baseline (B). Error bars represent SEM. *p &#60; 0.05, ***p &#60; 0.001. This figure has been modified from Smith et al.13. <a href="https://www.jove.com/files/ftp_upload/60026/60026fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60026/60026fig4.jpg" /> 
Figure 4: Evidence that the combination of stress and retrieval practice improved item memory (A) but not source memory (B). Error bars represent SEM. *p &#60; 0.05. This figure has been modified from Smith et al.13. <a href="https://www.jove.com/files/ftp_upload/60026/60026fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using Practice Testing, Public Speaking, and Source Monitoring to Examine the Influences of Learning Strategies and Stress on Episodic Memory at JoVE.com

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

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 ...

using-practice-testing-public-speaking-source-monitoring-to-examine

Research

JoVE Journal

Behavior

7.8K Views.  Tufts University. The present experiment combined three experimental procedures — a retrieval-practice learning manipulation, a list-discrimination task, and a stress-induction technique — to examine the influences of different learning strategies and acute stress on multiple measures of episodic memory.

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

Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.&#160;bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.&#160;distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.&#160;random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.&#160;shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g.&#160;latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land ...

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 ...

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

Using Wavelet Entropy to Demonstrate how Mindfulness Practice Increases Coordination between Irregular Cerebral and Cardiac Activities

In both the East and West, traditional teachings say that the mind and heart are somehow closely correlated, especially during spiritual practice. One difficulty in proving this objectively is that the natures of brain and heart activities are quite different. In this paper, we propose a methodology that uses wavelet entropy to measure the chaotic levels of both electroencephalogram (EEG) and electrocardiogram (ECG) data and show how this may be used to explore the potential coordination between the mind and heart under different experimental conditions. Furthermore, Statistical Parametric Mapping (SPM) was used to identify the brain regions in which the EEG wavelet entropy was the most affected by the experimental conditions. As an illustration, the EEG and ECG were recorded under two different conditions (normal rest and mindful breathing) at the beginning of an 8-week standard Mindfulness-based Stress Reduction (MBSR) training course (pretest) and after the course (posttest). Using the proposed method, the results consistently showed that the wavelet entropy of the brain EEG decreased during the MBSR mindful breathing state as compared to that during the closed-eye resting state. Similarly, a lower wavelet entropy of heartrate was found during MBSR mindful breathing. However, no difference in wavelet entropy during MBSR mindful breathing was found between the pretest and posttest. No correlation was observed between the entropy of brain waves and the entropy of heartrate during normal rest in all participants, whereas a significant correlation was observed during MBSR mindful breathing. Additionally, the most well-correlated brain regions were located in the central areas of the brain. This study provides a methodology for the establishment of evidence that mindfulness practice (i.e., mindful breathing) may increase the coordination between mind and heart activities.

This manuscript describes how to use the wavelet entropy index to analyze high-density electroencephalography (EEG) and electrocardiography (ECG) data. We show that the irregularity of cerebral and cardiac activities became more coordinated during mindfulness-based stress reduction practice.

In both the East and West, traditional teachings say that the mind and heart are somehow closely correlated, especially during spiritual practice. One ...

Novel Object Recognition Test for the Investigation of Learning and Memory in Mice

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is fairly simple and can be completed over 3 days: habituation day, training day, and testing day. During training, the mouse is allowed to explore 2 identical objects. On test day, one of the training objects is replaced with a novel object. Because mice have an innate preference for novelty, if the mouse recognizes the familiar object, it will spend most of its time at the novel object. Due to this innate preference, there is no need for positive or negative reinforcement or long training schedules. Additionally, the ORT can also be modified for numerous applications. The retention interval can be shortened to examine short-term memory, or lengthened to probe long-term memory. Pharmacological intervention can be used at various times prior to training, after training, or prior to recall to investigate different phases of learning (i.e., acquisition, early or late consolidation, or recall). Overall, the ORT is a relatively low-stress, efficient test for memory in mice, and is appropriate for the detection of neuropsychological changes following pharmacological, biological, or genetic manipulations.

The object recognition test (ORT) is a simple and efficient assay for evaluating learning and memory in mice. The methodology is described below.

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is ...

Reducing State Anxiety Using Working Memory Maintenance

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working Memory (WM) and the threat of shock paradigms. In the Sternberg WM paradigm, subjects are required to maintain a series of letters in the WM for a brief interval and respond by identifying whether the position of a given letter in the series matches a numerical prompt. In the threat of shock paradigm, subjects are exposed to alternating blocks where they are either at risk of receiving unpredictable presentations of a mild electric shock or are safe from the shock. Anxiety is probed throughout the safe and threat blocks using the acoustic startle reflex, which is potentiated under threat (Anxiety-Potentiated Startle (APS)). By conducting the Sternberg WM paradigm during the threat of shock and probing the startle response during either the WM maintenance interval or the intertrial interval, it is possible to determine the effect of WM maintenance on APS.

This protocol demonstrates how to measure anxiety-potentiated startle during the Sternberg Working Memory paradigm.

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working ...

The (Spatial) Memory Game: Testing the Relationship Between Spatial Language, Object Knowledge, and Spatial Cognition

The memory game paradigm is a behavioral procedure to explore the relationship between language, spatial memory, and object knowledge. Using two different versions of the paradigm, spatial language use and memory for object location are tested under different, experimentally manipulated conditions. This allows us to tease apart proposed models explaining the influence of object knowledge on spatial language (e.g., spatial demonstratives), and spatial memory, as well as understanding the parameters that affect demonstrative choice and spatial memory more broadly. Key to the development of the method was the need to collect data on language use (e.g., spatial demonstratives: &#34;this/that&#34;) and spatial memory data under strictly controlled conditions, while retaining a degree of ecological validity. The language version (section 3.1) of the memory game tests how conditions affect language use. Participants refer verbally to objects placed at different locations (e.g., using spatial demonstratives: &#34;this/that red circle&#34;). Different parameters can be experimentally manipulated: the distance from the participant, the position of a conspecific, and for example whether the participant owns, knows, or sees the object while referring to it. The same parameters can be manipulated in the memory version of the memory game (section 3.2). This version tests the effects of the different conditions on object-location memory. Following object placement, participants get 10 seconds to memorize the object&#39;s location. After the object and location cues are removed, participants verbally direct the experimenter to move a stick to indicate where the object was. The difference between the memorized and the actual location shows the direction and strength of the memory error, allowing comparisons between the influences of the respective parameters.

The Spatial Memory Game: Testing the Relationship Between Spatial Language, Object Knowledge, and Spatial Cognition

We present a protocol to explore the relationship between spatial language production, spatial memory, and object knowledge. The procedure allows experimental manipulation of, and control over, conditions of object knowledge, language at instruction, and physical location, thus teasing apart cognitive and linguistic models describing interactions between these variables.

The memory game paradigm is a behavioral procedure to explore the relationship between language, spatial memory, and object knowledge. Using two different ...

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

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

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

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

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the appearance and behavior of visual stimuli, compared to manipulating live animals. Here, we present the FishSim Animation Toolchain, a software framework developed to provide researchers with an easy-to-use method for implementing 3D computer animations in behavioral experiments with fish. The toolchain offers templates to create virtual 3D stimuli of five different fish species. Stimuli are customizable in both appearance and size, based on photographs taken of live fish. Multiple stimuli can be animated by recording swimming paths in a virtual environment using a video game controller. To increase standardization of the simulated behavior, the prerecorded swimming path may be replayed with different stimuli. Multiple animations can later be organized into playlists and presented on monitors during experiments with live fish.
In a case study with sailfin mollies (Poecilia latipinna), we provide a protocol on how to conduct a mate-choice copying experiment with FishSim. We utilized this method to create and animate virtual males and virtual model females, and then presented these to live focal females in a binary choice experiment. Our results demonstrate that computer animation may be used to simulate virtual fish in a mate-choice copying experiment to investigate the role of female gravid spots as an indication of quality for a model female in mate-choice copying.
Applying this method is not limited to mate-choice copying experiments but can be used in various experimental designs. Still, its usability depends on the visual capabilities of the study species and first needs validation. Overall, computer animations offer a high degree of control and standardization in experiments and bear the potential to &#39;reduce&#39; and &#39;replace&#39; live stimulus animals as well as to &#39;refine&#39; experimental procedures.

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Using the novel FishSim Animation Toolchain, we present a protocol for non-invasive visual manipulation of public information in the context of mate-choice copying in sailfin mollies. FishSim Animation Toolchain provides an easy-to-use framework for the design, animation and presentation of computer-animated fish stimuli for behavioral experiments with live test fish.

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the ...

Using the Visual World Paradigm to Study Sentence Comprehension in Mandarin-Speaking Children with Autism

Sentence comprehension relies on the ability to rapidly integrate different types of linguistic and non-linguistic information. However, there is currently a paucity of research exploring how preschool children with autism understand sentences using different types of cues. The mechanisms underlying sentence comprehension remains largely unclear. The present study presents a protocol to examine the sentence comprehension abilities of preschool children with autism. More specifically, a visual world paradigm of eye-tracking is used to explore the moment-to-moment sentence comprehension in the children. The paradigm has multiple advantages. First, it is sensitive to the time course of sentence comprehension and thus can provide rich information about how sentence comprehension unfolds over time. Second, it requires minimal task and communication demands, so it is ideal for testing children with autism. To further minimize the computational burden of children, the present study measures eye movements that arise as automatic responses to linguistic input rather than measuring eye movements that accompany conscious responses to spoken instructions.

We present a protocol to examine the use of morphological cues during real-time sentence comprehension by children with autism.