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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