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 conditions for light (12:12 light/dark schedule with lights on at 10:00 h), room temperature (25.0 &#177; 0.5 &#176;C), humidity (55 &#177; 10%), and ventilation (10 times/h), and animals had ad libitum access to food (Oriental Yeast Co. Ltd., Tokyo, Japan) and water. The animals were kept in a standard rat cage (W410 x D250 x H200 mm, Toyoriko Co., Ltd., Aichi, Japan) with paper bedding (alpha-dri, LSG Corporatin, Tokyo, Japan).
1. Elevated Plus Maze Test
<ol>
	<li>Set up the elevated plus maze apparatus in a customized container (W1,500 &#215; D1,500 &#215; H2,000 mm) (Figure 1A) with a plus-shaped metal framework 500 mm above the floor to support the run way arms (Figure 1B), lighting in the container ceiling to illuminate the surface of the maze to the required light intensity (Figure 1C), and a digital video camera in the center ceiling to enable live video monitoring of the behavioral tests (Figure 1C).</li>
	<li>Fix the arms (L500 x W100 mm) on the plus-shaped framework, and then fix the square central platform (W100 x D100 mm) at the center of the four arms (Figure 1D - F). 
	NOTE: In our laboratory, we have two types of open arms (L500 x W100 mm) and two types of closed arms (L500 x W100 x H450 mm) (Figure 1D). The closed arms have either transparent or opaque walls (Figure 1D), and the open arms are designed with or without a short ledge (H5 mm, Figure 1E). The square central platform (W100 &#215; D100 mm) is placed at center of the four arms (Figure 1D - F). Thus, the following four combinations of arms can be prepared according to experimental purpose: open arms with ledges + closed arms with transparent walls (Ledges/Transparent), open arms with ledges + closed arms with opaque walls (Ledges/Opaque), open arms without ledges + closed arms with transparent walls (No-Ledges/Transparent), and open arms without ledges + closed arms with opaque walls (No-Ledges/Opaque). The surface of the arm runway is black, to enable better animal tracing by the automated behavior analysis software, as described in&#160;Section 2, Behavioral Analysis.</li>
	<li>Adjust lighting intensity on the surface of the distal end part of each arm and central platform to approximately 100 Lux.</li>
	<li>Before each test, clean the entire apparatus with 70% ethanol solution and wipe with distilled water.</li>
	<li>Click the &#34;record button&#34; on the hard disk recorder to start the digital video camera that will record the rat&#39;s behavior for the video imaging analysis described in Section 2, Behavioral Analysis.</li>
	<li>Place an animal on the central platform facing an open arm and allow free exploration on the apparatus for 5 min.</li>
	<li>Click the &#34;stop button&#34; on the hard disk recorder to finish the recording.</li>
	<li>Count the number of feces and urinations after returning the tested animal to the animal housing room.</li>
	<li>Repeat from step 1.4 until the end of the experiment. 
	NOTE: A dry test run is always carried out before starting each experiment to check that all the experimental settings are correctly set up before any experimental animals are exposed to the experimental conditions. Otherwise, some animals, especially the first animal may experience different environmental conditions (e.g., noises possibly induced by the experimenter during manipulation and/or animal cues possibly left by the previously tested animal). 
	NOTE: The experimenters are near the container when an animal is being tested. Thus, even though the experimenters are not visible the rat may still be able to sense them via smell and/or sound.</li>
</ol>
2. Behavioral Analysis
Note: Behavioral analysis is performed by manual observation and automated analysis software.
<ol>
	<li>Manual observation
	<ol>
		<li>After the animal is placed on the ledge, observe the following: number of entries (an entry onto an arm is defined as any instance when all 4 paws are on the arm), time spent on each arm, and number of stretched-attending postures facing an open arm, while backing off the central platform towards a closed arm.</li>
		<li>Measure duration and&#160;frequency of the behaviors defined above by stopwatch and count for 5 min, starting from when the doors of the container are closed.</li>
	</ol>
	</li>
	<li>Automated analysis
	<ol>
		<li>Using the available software program, record the following: distance traveled, time spent in different positions on open arms (proximal, middle, and distal segments from the central platform), and a minute-by-minute analysis of the time spent on open and closed arms.</li>
		<li>Click the &#34;start button&#34; of the automated analysis software at the same time as the manual observation begins (the recording will stop automatically if recording duration is set). 
		NOTE: The behavioral analysis software used in this study tracks the animal via its contrast with the background field; the animal, which is assigned a fixed size, is identified as the brighter object and the locomotion of its center point can thus be used for animal tracking.</li>
	</ol>
	</li>
</ol>

<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>

The authors have nothing to disclose.

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 test animal anxiety in rodents4, but although the basic structures are usually consistent (e.g., elevated 500 mm above the floor with a plus shape), various arm designs are often used. Originally, the apparatus consisted of open arms without ledges, and closed arms with wooden opaque walls. However, more recently open arms have been developed with ledges to prevent animals falling off the apparatus, and closed arms have also been developed with transparent walls to facilitate observation of behavior on the closed arms. However, there have been no reports that addressed the combined effects of these arm features on the experimental outcomes in the elevated plus maze test. As shown by our results, the presence of open arm ledges and the transparency of the closed arm walls decreased avoidance of open arm exploration in rats (Figure 2), which may decrease the detection sensitivity of the anxiolytic effect of diazepam (Figure 3). The Ledges/Transparent apparatus appeared to encourage greater exploration on the open arms by vehicle treated rats (Figure 3). This reduced behavioral discrimination in open arm exploration activity between vehicle and diazepam treated rats, thus making it more difficult to detect differences in anxiety levels. Therefore, the lower sensitivity of Ledges/Transparent apparatus may mean that more animals would need to be tested to demonstrate significant treatment effects compared with using No-Ledges/opaque apparatus.
In our protocol, the duration and number of entries onto each arm was manually measured by well-trained experimenters. This procedure enables accurate measurement of arm entries, defined as all 4 paws placed on an arm. Although it is true that automated animal tracking systems are available for some behavioral parameters, such as traveling distance, in our experience they cannot always accurately distinguish between entries and non-entries onto arms, especially when animals perform stretched-attending postures near the border between the central platform and an arm. Thus, we recommend using a combination of both manual and automated observation systems, based on their respective strengths for each behavioral parameter.
There are some other protocol differences between laboratories. For example, some researchers place animals on the central platform facing a closed arm13,14, whereas others place the rats facing an open arm15,16. In addition, some researchers test behavior immediately after taking rats from their home-cage, whereas others allow the rats some time to acclimatize to the behavior testing room before starting the test. With respect to former case, in preliminary tests we experienced how the outcomes differed depending on which arm the rat initially faced; there was an increased incidence of entering onto the arm that the rat was placed facing. Also, when the rats were placed facing a closed arm, some animals did not move out of that closed arm after the first entry. In such a case, it is difficult to distinguish whether the rats experience conflict between the open and closed arms or that they merely prefer to be in a closed place without ever recognizing the presence of the open arms. Although in our study we chose to place the rats facing an open arm, to ensure a high incidence of being under conflict, we recommend that further studies be conducted to ensure that the effect of this protocol difference on experimental outcomes is properly investigated.
The measurements of exploration of the distal segment of open arms and minute-by-minute analysis provided extra detailed information about rat anxiety-like behavior. These measurements helped to show the differences in detection sensitivity between the different arm designs. Despite this, reported results of anxiety research using the elevated plus maze test often do not report these types of detailed measurements13,17,18. Therefore, we highly recommend that researchers also report further information about rat exploratory activities, including distal exploration, minute-by-minute analysis, and also stretched-attending posture.
In conclusion, our findings suggest that different types of apparatuses may have their own characteristics. From these tests, we have demonstrated that No-ledges/Opaque apparatus can better detect the effect of drugs potentially having an anxiolytic effect. In contrast, it is conceivable that Ledges/Transparent apparatus may have a similar advantage when testing potential anxiogenic drugs. To date, behavioral tests have been used not only for drug screening, but also for phenotyping assays in mutant animals. Indeed, advanced genome editing techniques allow rapid production of mutant animals. Therefore, it is very important to develop testing procedures with better precision and sensitivity to better detect the variations in behavior. This report demonstrates an improved application of the elevated plus test for animal anxiety research based on the expected functions of the drug or target genes. Thus, we believe that our efforts will help to advance research employing the elevated plus maze.

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)12, suggesting that the presence of ledges increases rat exploratory behavior on open arms. Rats significantly showed less stretched-attending posture on the apparatus that had closed arms with transparent walls than on apparatus that had closed arms with opaque walls (F[1,60]= 4.03, P = 0.0492) (Figure 2B), suggesting that transparent closed arm walls decreased anxiety-like behavior in rats. Rats spent more time on the distal segment of open arms on the apparatus that had transparent closed arm walls than on apparatus with opaque closed arm walls (F[1,60] = 8.00, P = 0.0983) (Figure 2C), suggesting that transparent walls may also decrease rat open arm exploratory behavior. Furthermore, open arm exploration on the distal segment was significantly lower in the No-ledges/Opaque apparatus compared to the Ledges/Transparent apparatus (Figure 2C). Taken together, these results indicate that detection sensitivity for anxiety-like behavior could differ depending on the types of arm design features used, and the No-ledges/Opaque apparatus and Ledges/Transparent apparatus may have a relatively large difference in the detection sensitivity.
To verify differences in detection sensitivity between different types of arm design, a further experiment was performed using the anxiolytic drug diazepam (Figures 3A-D). In brief, the No-Ledges/Opaque apparatus detected the effect of high-dose diazepam treatment on the time spent in the open arms, whereas the Ledges/Transparent apparatus did not (Figure 3A-B). Similar results were also found for the number of entries into open arms12 (data not shown). The lack of any significant effect on the Ledges/Transparent apparatus may have been due to the fact that the basal activity (vehicle treatment) on this apparatus was higher than on the No-Ledges/Opaque apparatus, but that open arm exploration never exceeded 33% of the time at any stage on either apparatus. Therefore, this narrower open arm exploration time range made it more difficult to distinguish the anxiolytic effect of diazepam on the Ledged/Transparent apparatus (Figure 3C-D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56428/56428fig1.jpg" /> 
Figure 1: Custom-designed equipment for elevated plus maze test. Panel (A): Elevated plus maze test container. The apparatus is placed in the black container, and a monitor next to the container projects live movie images recorded during the experiment. Panel (B): Plus maze framework in the container that supports fitted arms. Entire apparatus is located 500 mm above the floor. Panel (C): Ceiling lighting that can be adjusted for intensity. A digital video camera (see arrow) surrounded by&#160;lighting records animal movements. Panel (D): Elevated plus maze arms and central platform that are fitted to framework; from left-to-right, opaque wall closed arm, no-ledge open arm, ledged open arm, central platform, transparent wall closed arm. Panel (E): Close-up view of open arms; from left-to-right, open arm without ledges, open arm with ledges (H5 mm). Panel (F): An example of a constructed maze (Ledges/Transparent apparatus). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56428/56428fig1large.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/56428/56428fig2.jpg" /> 
Figure 2: Open arm ledges reduce anxiety-like behavior, and transparent walls partially decrease anxiety-like behavior. Male rats underwent the elevated plus maze test with different combinations of arm structures (Ledges/Transparent, Ledges/Opaque, No-ledges/Transparent, and No-ledges/Opaque). Testing order was normalized across the experimental groups (each combination of apparatus type). This procedure normalizes unexpected effects that could have occurred due to the time of day of each test. Panel (A): Time spent on open arms; Panel (B): Number of stretched-attending postures; Panel (C): Time spent in the distal segment of open arms. Solid and dashed lines indicate significant main effects of open and closed arm structures, respectively (Two-way ANOVA, #P &#60; 0.1, * P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.001). Different letters beside the bars in Panel (C) indicate a significant difference (Bonferroni multiple comparison test, a &#60; b, P &#60; 0.0125). The data were expressed as mean with SEM (n = 16 per experimental group). These figures have been modified from our previous report12. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56428/56428fig2large.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/56428/56428fig3.jpg" /> 
Figure 3: No-Ledges/Opaque apparatus shows higher detection sensitivity of anxiolytic effect of diazepam. Male rats underwent the elevated plus maze test, with either No-Ledges/Opaque or Ledges/Transparent apparatus, 30 min after being treated with either vehicle (0 mg/kg), low (0.25 mg/kg), or high (0.5 mg/kg) doses of the anxiolytic drug diazepam (intraperitoneal injection). Diazepam solution was dissolved in 0.9% normal saline solution with a surface-activating agent. In this experiment, the testing order of the three drug treatment groups and two types of apparatus design were randomized to account for any effects that may occur due to the time of day of each test. Panels (A &#38; B): Total percent time (5 min) spent on open arms of Ledges/Transparent and No-ledges/Opaque apparatus, respectively. Panels (C &#38; D): Minute-by-minute percent time spent on open arms of Ledges/Transparent and No-ledges/Opaque apparatus, respectively. Different letters beside bars in Panel (B) indicate a significant difference between treatments (Bonferroni multiple comparison test, a &#60; b, P &#60; 0.0167). Solid and dashed lines in Panels (C &#38; D) indicate significant main effects of the time-lapse and diazepam treatments, respectively (repeated measures ANOVA, *P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.001). The data were expressed as mean with SEM. Numbers in the columns indicate animals that completed the experiment (numbers in parenthesis indicate animals falling off the apparatus). These figures have been modified from our previous report12. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56428/56428fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

testing-animal-anxiety-rats-effects-open-arm-ledges-closed-arm-wall

Nanyang Technological University

Research

JoVE Journal

Behavior

13.4K Views.  Meiji University. 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. 

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

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 Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

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 ...