Name: testing animal anxiety in rats: effects of open arm ledges and closed arm wall transparency in elevated plus maze test
Uploaded: 2018-06-29T12:30:00
Description: Watch this Scientific Journal Video about Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test at JoVE.com

The authors wish to thank Mr. Akihiro Hirano for his assistance in producing the customized apparatus. The authors would like to thank Mr. Kosuke Takahashi and Mr. Shingo Nakajima for their technical assistance. This work was supported by Research on Risk of Chemical Substances, Health and Laboratory Sciences Research Grants, Ministry of Health, Labour and Welfare, Japan (H25-KAGAKU-IPPANN-003) and Research Project Grant A by Institute of Science and Technology Meiji University, Japan.

All experimental procedures described here have been approved by the Institutional Animal Care and Use Committee of Meiji University (IACUC 14-0002(1)). Animals used in the representative results were prepared as follows: Male Sprague-Dawley rats were purchased from a laboratory animal company and housed in groups of 4 animals per standard rat cage (W200 &#215; D410 &#215; H250 mm) for at least one week before the start of the behavioral experiment.&#160;The animal housing room was maintained at standard ambient conditio...

<ol>
	<li>Nestler, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Brain's+Reward+Circuitry+in+Depression:+Transcriptional+Mechanisms.">Role of the Brain's Reward Circuitry in Depression: Transcriptional Mechanisms.</a> Int Rev Neurobiol. 124, 151-170 (2015).</li><li>Crabbe, J. C., Wahlsten, D., Dudek, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+mouse+behavior:+interactions+with+laboratory+environment.">Genetics of mouse behavior: interactions with laboratory environment.</a> Science. 284 (5420), 1670-1672 (1999).</li><li>Handley, S. L., Mithani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+alpha-adrenoceptor+agonists+and+antagonists+in+a+maze-exploration+model+of+'fear'-motivated+behaviour.">Effects of alpha-adrenoceptor agonists and antagonists in a maze-exploration model of 'fear'-motivated behaviour.</a> Naunyn Schmiedebergs Arch Pharmacol. 327 (1), 1-5 (1984).</li><li>Pellow, S., Chopin, P., File, S. E., Briley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+open:closed+arm+entries+in+an+elevated+plus-maze+as+a+measure+of+anxiety+in+the+rat.">Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat.</a> J Neurosci Methods. 14 (3), 149-167 (1985).</li><li>Violle, N., Balandras, F., Le Roux, Y., Desor, D., Schroeder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+illumination,+closed+wall+transparency+and/or+extramaze+space+influence+both+baseline+anxiety+and+response+to+diazepam+in+the+rat+elevated+plus-maze.">Variations in illumination, closed wall transparency and/or extramaze space influence both baseline anxiety and response to diazepam in the rat elevated plus-maze.</a> Behav Brain Res. 203 (1), 35-42 (2009).</li><li>Hogg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+validity+and+variability+of+the+elevated+plus-maze+as+an+animal+model+of+anxiety.">A review of the validity and variability of the elevated plus-maze as an animal model of anxiety.</a> Pharmacol Biochem Behav. 54 (1), 21-30 (1996).</li><li>Martinez, J. C., Cardenas, F., Lamprea, M., Morato, 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+role+of+vision+and+proprioception+in+the+aversion+of+rats+to+the+open+arms+of+an+elevated+plus-maze.">The role of vision and proprioception in the aversion of rats to the open arms of an elevated plus-maze.</a> Behav Processes. 60 (1), 15-26 (2002).</li><li>Anseloni, V. Z., Brandao, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethopharmacological+analysis+of+behaviour+of+rats+using+variations+of+the+elevated+plus-maze.">Ethopharmacological analysis of behaviour of rats using variations of the elevated plus-maze.</a> Behav Pharmacol. 8 (6-7), 533-540 (1997).</li><li>Anseloni, V. Z., Motta, V., Lima, G., Brandao, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+pharmacological+validation+of+the+elevated+plus+maze+constructed+with+transparent+walls.">Behavioral and pharmacological validation of the elevated plus maze constructed with transparent walls.</a> Braz J Med Biol Res. 28 (5), 597-601 (1995).</li><li>Hagenbuch, N., Feldon, J., Yee, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+elevated+plus-maze+test+with+opaque+or+transparent+walls+in+the+detection+of+mouse+strain+differences+and+the+anxiolytic+effects+of+diazepam.">Use of the elevated plus-maze test with opaque or transparent walls in the detection of mouse strain differences and the anxiolytic effects of diazepam.</a> Behav Pharmacol. 17 (1), 31-41 (2006).</li><li>Fernandes, C., File, S. 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+influence+of+open+arm+ledges+and+maze+experience+in+the+elevated+plus-maze.">The influence of open arm ledges and maze experience in the elevated plus-maze.</a> Pharmacol Biochem Behav. 54 (1), 31-40 (1996).</li><li>Horii, Y., Kawaguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+detection+sensitivity+of+anxiolytic+effects+of+diazepam+by+ledge-free+open+arm+with+opaque+walled+closed+arm+elevated+plus+maze+in+male+rats.">Higher detection sensitivity of anxiolytic effects of diazepam by ledge-free open arm with opaque walled closed arm elevated plus maze in male rats.</a> Behav Brain Res. 294, 131-140 (2015).</li><li>Santos, R. O., 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=Evaluation+of+the+effect+of+acute+sibutramine+in+female+rats+in+the+elevated+T-maze+and+elevated+plus-maze+tests.">Evaluation of the effect of acute sibutramine in female rats in the elevated T-maze and elevated plus-maze tests.</a> Basic Clin Pharmacol Toxicol. 114 (2), 181-187 (2014).</li><li>Lynn, D. A., Brown, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ontogeny+of+anxiety-like+behavior+in+rats+from+adolescence+to+adulthood.">The ontogeny of anxiety-like behavior in rats from adolescence to adulthood.</a> Dev Psychobiol. 52 (8), 731-739 (2010).</li><li>Horii, Y., 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=Male+Hatano+high-avoidance+rats+show+high+avoidance+and+high+anxiety-like+behaviors+as+compared+with+male+low-avoidance+rats.">Male Hatano high-avoidance rats show high avoidance and high anxiety-like behaviors as compared with male low-avoidance rats.</a> Exp Anim. 61 (5), 517-524 (2012).</li><li>Walf, A. A., Frye, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+elevated+plus+maze+as+an+assay+of+anxiety-related+behavior+in+rodents.">The use of the elevated plus maze as an assay of anxiety-related behavior in rodents.</a> Nat Protoc. 2 (2), 322-328 (2007).</li><li>Yang, Y., 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=Behavioral+and+pharmacological+investigation+of+anxiety+and+maternal+responsiveness+of+postpartum+female+rats+in+a+pup+elevated+plus+maze.">Behavioral and pharmacological investigation of anxiety and maternal responsiveness of postpartum female rats in a pup elevated plus maze.</a> Behav Brain Res. 292, 414-427 (2015).</li><li>Albani, S. H., 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=Behavior+in+the+elevated+plus+maze+is+differentially+affected+by+testing+conditions+in+rats+under+and+over+three+weeks+of+age.">Behavior in the elevated plus maze is differentially affected by testing conditions in rats under and over three weeks of age.</a> Front Behav Neurosci. 9, 31 (2015).</li></ol>

As the number of patients suffering from psychiatric disorders, including anxiety-disorder, increases, it is important to develop cutting-edge studies to improve understanding of the basic mechanism of anxiety. To achieve this, it is also important to optimize the experimental procedures. Therefore, to improve the application of the elevated plus maze test, we have demonstrated the effect of arm features on experimental outcomes using rats.
The elevated plus maze was originally established to ...

The number of patients suffering from mood disorders, including anxiety-disorder and depression, is increasing. Although the development of these disorders involves both genetic and environmental factors, the detailed pathogenic mechanisms are not yet fully understood1. Therefore, research needs to focus on uncovering the underlying mechanisms of mood disorders, as well as the development of effective drugs that have fewer side effects. 
Behavioral tests for laboratory animals are useful for investigating brain higher function, including emotion. As animal behavior is highly variable, experimental outcomes often differ between laboratories, even if an identical inbred strain was used2. Thus, researchers also need to understand the basic features of the experimental devices they adopt in order to provide reliable, precise outcomes in the laboratory.
The elevated plus maze test was originally established to examine emotional reactivity in rodents3,4. The entire apparatus is usually located 500 mm above the floor, and consists of two open and two closed arms that make a plus shape. Measurements of arm exploration (including duration and frequency of entries on each arm), distance traveled, and specified behaviors such as stretched-attending posture of subjected animals are recorded during observational periods. By these measures, animal anxiety is estimated as an experimental outcome. 
The elevated plus maze test is used throughout the world, but not all laboratories use the same apparatus design5,6, and these differences may affect the experimental outcomes. For example, differences include the presence or absence of ledges along the open arms and/or the use of transparent or opaque walls for closed arms5,6. The ledges along open arms and the use of transparent walls for closed arms were developed to prevent animals falling off the apparatus7, and to facilitate behavioral observations inside the closed arms, respectively8,9. Although there have been some studies that examined how individual arm structure differences may affect the ability to detect animal anxiety1,2,5,10,11, the combined effects of open (with/without short ledge) and closed (transparent/opaque walls) arm features on animal behavior has not yet been investigated. 
In a recent report, we discussed the effect of these combinations of arm designs on anxiety-like behavior in rats12. In brief, open arm ledges significantly increased anxiety-like behavior in rats, while transparent arms may also have decreased this behavior. Furthermore, we verified higher detection sensitivity of the effect of an anxiolytic drug in a combination of no-ledged open arm + opaque walled closed arm compared with a combination of ledged open arm + transparent walled closed arm. In this report, we will introduce our protocol for the elevated plus maze test, together with discussion of the key results from the previous report. We believe this report will provide useful information for those who have employed or who plan to use the elevated plus maze in their studies.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Customized container (W1500 &#215; D1500 &#215; H2000 mm)</td>
			<td style="width:71px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:167px;">Handmade by Mr. Akihiro Hirano</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LED lighting</td>
			<td style="width:71px;">NA</td>
			<td style="width:119px;">NA</td>
			<td>Handmade by Mr. Akihiro Hirano</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Elevated plus maze apparatus</td>
			<td style="width:71px;">NA</td>
			<td style="width:119px;">NA</td>
			<td>Handmade by Mr. Akihiro Hirano</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digital video camera</td>
			<td style="width:71px;">Watec</td>
			<td style="width:119px;">WAT-250D2</td>
			<td>Any suitable video camera can be attached to a hard disk recorder for recording tracking profiles.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hard disk recorder</td>
			<td style="width:71px;">TOSHIBA</td>
			<td style="width:119px;">RD-R200</td>
			<td>Any suitable hard disk recorder can be attached to the digital video camera.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Sprague-Dawley rats</td>
			<td style="width:71px;">Charles River Laboratories Japan, Inc.</td>
			<td style="width:119px;">NA</td>
			<td>Any suitable rats can be used for your study</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard rat cage</td>
			<td style="width:71px;">Toyoriko</td>
			<td style="width:119px;">TP-105</td>
			<td>Any suitable rat cage be used for your study</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Instrument to measure light intensity</td>
			<td style="width:71px;">AS ONE</td>
			<td style="width:119px;">1-5818-02</td>
			<td>Any suitable instrument to measure light intensity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EthoVision XT version 8.0</td>
			<td style="width:71px;">Noldus</td>
			<td style="width:119px;">NA</td>
			<td>Any suitable software to measure animal behavior</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Diazepam</td>
			<td style="width:71px;">Taiyo Pharmaceutical Industry</td>
			<td style="width:119px;">1124402A2061</td>
			<td>Any suitable drug can be used for your study</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Tween 80</td>
			<td style="width:71px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;">9005-65-6</td>
			<td>Any suitable drug can be used for your study</td>
		</tr>
	</tbody>
</table>

testing animal anxiety in rats: effects of open arm ledges and closed arm wall transparency in elevated plus maze test

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral science, conflicting outcomes are often provided from different laboratories. To identify reasons for the different outcomes, we previously focused on arm features, which differ between laboratories, most notably the presence/absence of ledges at the sides of open arms and the transparency/opaqueness of closed arm walls. In a previous report, we used a custom designed container to compare rat behavior on different combinations of open and closed arm designs under otherwise identical experimental conditions, and showed that differences in arm features interfere with experimental outcomes. In brief, open arm ledges significantly increased anxiety-like behavior in rats, while transparent arms may also have decreased this behavior. Furthermore, we verified a higher detection sensitivity of the effect of an anxiolytic drug in a combination of no-ledged open arm + opaque walled closed arm compared with a combination of ledged open arm + transparent walled closed arm. In this report, we introduce our protocol for the elevated plus maze test, together with discussion of the key results from the previous report and our experimental experience. We believe this report will provide useful information for researchers who have employed or who plan to use the elevated plus maze in their studies.

In the previous study, we compared the combined effects of open arm ledges and transparency of closed arm walls on the exploratory behaviors on the maze12. Rats significantly spent much less time on open arms without ledges than on open arms with ledges (F[1, 60] = 13.49, P = 0.0005) (Figure 2A). Similar results were also found for the number of entries into open arms (F[1, 60] = 11.17, P &#60; 0.0014)<sup class="xref...

Watch this Scientific Journal Video about Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test at JoVE.com

Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test

Rats' anxiety-like behavior tested by the elevated plus maze varies highly depending on the testing environment. Here we show how apparatus settings, especially arm structure, affect behavior on the maze, and we explain a reliable protocol to detect effects of anxiolytic drugs. This report will contribute to anxiety research.

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral ...

The overall goal of this experiment is to measure anxiety in rats, using the appropriate elevated plus maze. The main finding of this study is that the detection sensitivity of the anxiolytic drug effect can change depending on the features of the elevated cross maze apparatus. This study can help researchers set up the maze apparatus in the laboratory, appropriate for their experimental objective, and ensure a great recording of data.Demonstrating the procedure will be a guest researcher from my laboratory. To begin set up the elevated plus maze apparatus in a customized closet container. The plus shaped metal framework of the maze should be elevated about half a meter from the floor.Maintain a consistent brightness throughout the maze at the required intensity. At the overhead center point mount a digital video camera to record and monitor the tests. Now fix the maze arms onto the framework.There are two types of open arms, either with or without 5MM ledges along the sides. There are also two types of closed arms. The walls can be translucent or opaque.Attach the square central platform between the arms. The central platform and maze arm runway surfaces are colored black for better animal tracking by the automated behavioral analysis. Now with arms in place adjust the lighting intensity, so there is approximately 100 lux at the surface of the distal end part of each arm, and at the central platform.Before each test clean the entire apparatus with 70%ethanol solution, followed by wiping it down with distilled water. Click the record button on the hard disk recorder, to start collecting images from the digital video camera. If the equipment is working then conduct the test.Start the test by placing the rat on the central platform, so that it is facing an open arm, and close the closet doors. Then allow free exploration on the apparatus for five minutes. The experimental outcome may vary, depending on whether the rat is appraised facing an open or crossed arm.It is better to always place rats, resting on open arm to prevent freezing, on first entry to the closed arm. After five minutes, stop the recording to end the test. Then return the rat to its cage, and count the number of feces and urination's in the maze before cleaning it.Always perform the behavioral analysis using both manual observation, and automated analysis software. From the video manually record the number of entries the rat makes into the arms of the maze. An entry is defined as any instance when all four paws are on the arm.Also record the time spent in each arm, and the number of stretched attending postures made while the rat is on the central platform facing towards an open arm, and backing into a closed arm. Perform these measurements using a stopwatch and a counter during the five minute test. To perform an automated analysis, open the video recording in a commercial software program.The software tracks the animal on the maze, using the contrast between the animal and the dark floor. First assign the animal to a contrast blob of a specific size and then assign the center of the blob as the tracking point. Next for the full five minute test, have the software record the distance traveled, the time spent in different positions on open arms, and the total time spent on open and closed arms.Sometimes the automated analysis cannot distinguish between entries and non entries into arms. Therefore, we recommend that manual observation is always carried out in addition to automated analysis. The results of rat exploratory behavior on the elevated plus maze with different open and closed arm designs, show that rats spend more time on open arms with ledges, than open arms without ledges.They also made more entries onto those arms, thus ledges likely increase exploratory behavior on open arms. Rats showed significantly less stretched-attending posture on closed arms with transparent walls, than on closed arms with opaque walls. Suggesting that transparent closed arm walls decreased anxiety like behavior.Rats also spent more time on the distal segments of the open arms when the closed arms had transparent walls, than when the closed arms had opaque walls. Furthermore, distal open arm exploration was significantly lower in mazes with no ledges and opaque arm combinations, than in ledges and transparent arm combinations. Therefore detection sensitivity maybe altered, depending on the combination of arm designs used.To verify differences in detection sensitivity between different types of arm design, a further experiment was performed using the anxiolytic drug Diazepam. I brief, the effect of high dose Diazepam treatment was detected in the opaque wall apparatus without ledges, but not in the transparent wall apparatus with ledges. While attempting this procedure, it's important to remember to treat all animals under the same experimental conditions, and to make sure that all animals are placed facing the same open or closed arm at the start of each test.After watching this video you should understand how to set up the elevated cross maze apparatus. It is important to remember that experimental outcomes can vary depending on arm design. So we recommend using appropriate arms for your experimental objectives.

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Research

JoVE Journal

Behavior

The Successive Alleys Test of Anxiety in Mice and Rats

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones. Handley, Mithani and File et al. performed the first studies on the plus-maze design we use today, and in 1987 Lister published a design for use with mice. 
Time spent on, and entries into, the open arms are an index of anxiety; the lower these indices, the more anxious the mouse is. Alternatively, a mouse that spends most of its time in the closed arms is classed as anxious. 
One of the problems of the plus-maze is that, while time spent on, and entries into, the open arms is a fairly unambiguous measure of anxiety, time in the central area is more difficult to interpret, although time spent here has been classified as &#x201C;decision making&#x201D;. In many tests central area time is a considerable part of the total test time.
Shepherd et al. produced an ingenious design to eliminate the central area, which they called the &#x201C;zero maze&#x201D;. However, although used by several groups, it has never been as widely adopted as the plus-maze. 
In the present article I describe a modification of the plus-maze design that not only eliminates the central area but also incorporates elements from other anxiety tests, such as the light-dark box and emergence tests. It is a linear series of four alleys, each having increasing anxiogenic properties. It has given similar results to the plus-maze in general. Although it may not be more sensitive than the plus-maze (more data is needed before a firm conclusion can be reached on this point), it provides a useful confirmation of plus-maze results which would be useful when, for example, only a single example of a mutant mouse was available, as, for example, in ENU-based mutagenesis programs.

The plus-maze measures anxiety-like behaviour in rodents. There are two opposite closed and two opposite open arms; anxious rodents avoid the open arms. The central area is neither completely open nor closed, so time spent here is ambiguous and difficult to interpret. Here a modification of the plus-maze protocol eliminating this area is described.

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones.  ...

An Alternative to the Traditional Cold Pressor Test: The Cold Pressor Arm Wrap

Recently research on the relationship between stress and cognition, emotion, and behavior has greatly increased. These advances have yielded insights into important questions ranging from the nature of stress&#39; influence on addiction1 to the role of stress in neural changes associated with alterations in decision-making2,3. As topics being examined by the field evolve, however, so too must the methodologies involved. In this article a practical and effective alternative to a classic stress induction technique, the cold pressor test (CPT), is presented: the cold pressor arm wrap (CPAW). CPT typically involves immersion of a participant&#39;s dominant hand in ice-cold water for a period of time4. The technique is associated with robust activation of the sympatho-adrenomedullary (SAM) axis (and release of catecholamines; e.g.&#160;adrenaline and noradrenaline) and mild-to-moderate activation of the hypothalamic-pituitary-adrenal (HPA) axis with associated glucocorticoid (e.g.&#160;cortisol) release. While CPT has been used in a wide range of studies, it can be impractical to apply in some research environments. For example use of water during, rather than prior to, magnetic resonance imaging (MRI) has the potential to damage sensitive and expensive equipment or interfere with acquisition of MRI signal. The CPAW is a practical and effective alternative to the traditional CPT. Composed of a versatile list of inexpensive and easily acquired components, CPAW makes use of MRI-safe gelpacs cooled to a temperature similar to CPT rather than actual water. Importantly CPAW is associated with levels of SAM and HPA activation comparable to CPT, and can easily be applied in a variety of research contexts. While it is important to maintain specific safety protocols when using the technique, these are easy to implement if planned for. Creation and use of the CPAW will be discussed.

The cold pressor test is a classic stress induction task used in a wide range of research examining the relationship between stress and cognition, emotion, and behavior. In this article, a practical and versatile alternative to the traditional cold pressor test is presented: the cold pressor arm wrap.

Recently research on the relationship between stress and cognition, emotion, and behavior has greatly increased. These advances have yielded insights into ...

Use of an Eight-arm Radial Water Maze to Assess Working and Reference Memory Following Neonatal Brain Injury

Working and reference memory are commonly assessed using the land based radial arm maze.&#160;However, this paradigm requires pretraining, food deprivation, and may introduce scent cue confounds. The eight-arm radial water maze is designed to evaluate reference and working memory performance simultaneously by requiring subjects to use extra-maze cues to locate escape platforms and remedies the limitations observed in land based radial arm maze designs. Specifically, subjects are required to avoid the arms previously used for escape during each testing day (working memory) as well as avoid the fixed arms, which never contain escape platforms (reference memory). Re-entries into arms that have already been used for escape during a testing session (and thus the escape platform has been removed) and re-entries into reference memory arms are indicative of working memory deficits. Alternatively, first entries into reference memory arms are indicative of reference memory deficits. We used this maze to compare performance of rats with neonatal brain injury and sham controls following induction of hypoxia-ischemia and show significant deficits in both working and reference memory after eleven days of testing. This protocol could be easily modified to examine many other models of learning impairment.

The eight-arm radial water maze is designed to evaluate reference and working memory performance simultaneously by requiring subjects to use extra-maze cues to locate escape platforms and remedies the limitations observed in land based radial arm maze designs.

Working and reference memory are commonly assessed using the land based radial arm maze.&#160;However, this paradigm requires pretraining, food ...

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 ...

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

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, ...

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how &#8220;stress&#8221; might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function1. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats2. Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for ...

An Objective and Child-friendly Assessment of Arm Function by Using a 3-D Sensor

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment of muscle functions is an essential and important, although challenging, prerequisite for successful clinical trials. Current clinical rating scales restrain the movement abnormalities to certain predefined coarse-grained individual items. The Kinect 3-D sensor has emerged as a low-cost and portable motion sensing technology used to capture and track people&#39;s movement in many medical and research fields. A novel approach using this 3-D sensor was developed and a game-like test was designed to objectively measure the upper limb function of patients with SMA. The prototype test targeted joint movement capability. While sitting in a virtual scene, the patient was instructed to extend, flex, and lift the whole arm in order to reach and place some objects. Both kinematic and spatiotemporal characteristics of upper limb movement were extracted and analyzed, e.g., elbow extension and flexion angles, hand velocity, and acceleration. The first study included a small cohort of 18 ambulant SMA patients and 19 age- and gender-matched healthy controls. A comprehensive analysis of arm movement was achieved; however, no significant difference between the groups were found due to the mismatch of patient&#39;s capability and the test difficulty. Based on this experience, a second version of the test consisting of a modified version of the first game with increased difficulties and a second game targeting muscle endurance were designed and implemented. The new test has not been conducted in any patient groups yet. Our work has demonstrated the potential capability of the 3-D sensor in assessing such muscle function and suggested an objective approach to complement the clinical rating scales.

An objective measure of muscle functions is challenging especially in children. Based on a commercially available digital 3-D sensor, a child-friendly gaming test was developed to assess upper limb function for clinical trials.

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment ...

Testing for Metacognitive Responding Using an Odor-based Delayed Match-to-Sample Test in Rats

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, numerous behavioral tasks have been created, many of which utilize an option to decline memory tests. To assess metamemory in rats, we utilized this decline-test option paradigm by adapting previous visual delayed-match-to-sample tests (DMTS)1,2 developed for primate species to an odor-based test suitable for rodents. First,&#160;rats are given a sample to remember by digging in a cup of scented sand. After a delay, the rat is presented with four distinctly scented cups, one of which contains the identical scent experienced during the sample; if this matching cup is selected, then the rat obtains a preferred, larger reward. Selection of any of the other three non-matching sand-filled scented cups results in no reward. Retention intervals are individually titrated such that subjects perform between 40 and 70% correct, therefore ensuring rats sometimes remember and sometimes forget the sample. Here, the operational definition of metamemory is the ability to distinguish between the presence and absence of memory through behavioral responding. Towards this end, on two-thirds of trials, a decline option is presented in addition to the four choice cups (choice trials). If the decline-test option- an unscented colored sand cup, is selected, the subject receives a smaller less-preferred reward and avoids the memory test. On the remaining third of trials, the decline-test option is not available (forced trials), causing subjects to guess the correct cup when the sample is forgotten. On choice tests, subjects that know when they remember should select the decline option when memory is weak rather than take the test and choose incorrectly. Therefore, significantly higher performance on chosen tests as compared to forced memory tests is indicative of the adaptive use of the decline-test response and metacognitive responding.

This protocol describes a method for investigating the possibility of metamemory, or memory awareness, in rodents. The odor-based delayed-matching-to-sample paradigm is a novel, ecologically-relevant behavioral test useful for determining the extent to which rodents can adaptively respond based on cognitively monitoring the strength of their memory states.

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, ...