This research was supported by Grants-in-Aid for Scientific Research of the Japan Society for the Promotion of Science (JSPS), Grant-in-Aid from Ministry of Education, Culture, Sports, Science and Technology in Japan, Grants-in-Aid from BIRD and CREST of Japan Science and Technology Agency, Grant-in-Aid from Neuroinformatics Japan Center (NIJC), RIKEN and by Grant-in-Aid for Scientific Research on Priority Areas-Integrative Brain Research (Shien)- from MEXT in Japan. We thank O'hara &amp; Co. and Mariko Hayashi for their assistance in creating this movie.

Protocol
<ol>
<li>The apparatus used for the elevated plus maze test is in the configuration of a + and comprises two open arms (25 x 5 x 0.5 cm) across from each other and perpendicular to two closed arms (25 x 5 x 16 cm) with a center platform (5 x 5 x 0.5 cm). The open arms have a very small (0.5 cm) wall to decrease the number of falls, whereas the closed arms have a high (16 cm) wall to enclose the arm. The entire apparatus is 50 cm above the floor (Ohara &amp; Co., Tokyo) and is placed in an empty circular tank (100 cm diameter, 35 cm tall; normally used for the Morris water maze task) to protect the mice that fall or attempt to escape during the experiment. The apparatus is made of plastic materials. The platform is white and the walls are transparent. There is a variation in materials and colors of the apparatus of elevated plus maze. </li>
<li>Mice are housed with a 12-h light/dark cycle (lights on at 7:00 AM), as previously described (Takao &amp; Miyakawa, 2006a). Behavioral testing is performed between 9:00 AM and 6:00 PM. All the experimental mice are transferred to the behavior testing room 30 min prior to beginning the first trial to habituate to the condition of the behavior testing room. The order of trials is counterbalanced across genotypes. There are two purposes of the test trial using a practice animal. One is to make sure that everything is fine with the recording system. Another is to keep the testing condition as uniform as possible. That is, the very first mouse in the entire session would experience somewhat different condition with others (i.e., no prior noises made by testing operations and no odor cues from previous trials) without such a practice trial. Animals are maintained according to the guidelines of the Animal Research Committee of Kyoto University. </li>
<li>The behavior testing room (170 x 210 x 200 cm, Ohara &amp; Co., Tokyo) is soundproof and the illumination level is maintained at 100 lux. A mouse is placed in the center area of the maze with its head directed toward a closed arm. The elevated plus maze test is recorded using a video camera attached to a computer, which is controlled by a remote device. The number of entries (an entry is defined as the center of mass of the mouse enters the arm) into each arm and the time spent in the open arms are recorded and these measurements serve as an index of anxiety-like behavior. </li>
<li>Mice are allowed to move freely about the maze for 10 min. Each mouse receives one trial in our test battery. The application used for acquiring and analyzing the behavioral data (Image EP) is based on the public domain Image J program (developed by Wayne Rasband at the National Institute of Mental Health and available at <a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</a>), which was modified by Tsuyoshi Miyakawa (available through O'Hara &amp; Co., Tokyo, Japan). </li>
<li>The distance traveled, the number of entries into each arm, the time spent in each arm, and the percent of entries into the open arms are calculated by the Image EP program. </li>
<li>After each trial, all arms and the center area are cleaned with super hypochlorous water, that is an efficient odor removal agent and has relatively weak odor of itself compared to other cleaning solutions, to prevent a bias based on olfactory cues. Thus we can conduct the tests under controlled condition regarding olfactory cues. </li>
</ol>

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<li>Seeger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((M2+muscarinic+acetylcholine+receptor+knockout+mice+show+deficits+in+behavioral+flexibility%2C+working+memory%2C+and+hippocampal+plasticity%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">M2 muscarinic acetylcholine receptor knockout mice show deficits in behavioral flexibility, working memory, and hippocampal plasticity.</a> J Neurosci. 24, 10117-10127 (2004).</li>
<li>Morishima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+cocaine+responsiveness+and+impaired+motor+coordination+in+metabotropic+glutamate+receptor+subtype+2+knockout+mice%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Enhanced cocaine responsiveness and impaired motor coordination in metabotropic glutamate receptor subtype 2 knockout mice.</a> Proc Natl Acad Sci USA. 102, 4170(2005).</li>
<li>Miyamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tight+junctions+in+Schwann+cells+of+peripheral+myelinated+axons%3A+a+lesson+from+claudin-19-deficient+mice%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Tight junctions in Schwann cells of peripheral myelinated axons: a lesson from claudin-19-deficient mice.</a> J Cell Biol. 169, 527-538 (2005).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NFAT+dysregulation+by+increased+dosage+of+DSCR1+and+DYRK1A+on+chromosome+21%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21.</a> Nature. 441, 595-600 (2006).</li>
<li>Hattori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enriched+environments+influence+depression-related+behavior+in+adult+mice+and+the+survival+of+newborn+cells+in+their+hippocampi%5BTitle%5D)+AND+%22Behav+Brain+Res%22%5BJournal%5D)">Enriched environments influence depression-related behavior in adult mice and the survival of newborn cells in their hippocampi.</a> Behav Brain Res. 180, 69-76 (2007).</li>
<li>Niemann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+ablation+of+NMDA+receptor+subunit+NR3B+in+mouse+reveals+motoneuronal+and+non-motoneuronal+phenotypes%5BTitle%5D)+AND+%22Europ+J+Neurosci%22%5BJournal%5D)">Genetic ablation of NMDA receptor subunit NR3B in mouse reveals motoneuronal and non-motoneuronal phenotypes.</a> Europ J Neurosci. 26, 1407-1420 (2007).</li>
<li>Sano, H., Nagai, Y., Miyakawa, T., Shigemoto, R., Yokoi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+social+interaction+in+mice+deficient+of+the+striatal+medium+spiny+neuron-specific+phosphodiesterase+10A2%5BTitle%5D)+AND+%22J+Neurochem%22%5BJournal%5D)">Increased social interaction in mice deficient of the striatal medium spiny neuron-specific phosphodiesterase 10A2.</a> J Neurochem. 105, 546-556 (2008).</li>
<li>Horii, Y., Yamasaki, N., Miyakawa, T., Shiosaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+anxiety-like+behavior+in+neuropsin+%28kallikrein-related+peptidase+8%29+gene-deficient+mice%5BTitle%5D)+AND+%22Behav+Neurosci%22%5BJournal%5D)">Increased anxiety-like behavior in neuropsin (kallikrein-related peptidase 8) gene-deficient mice.</a> Behav Neurosci. 122, 498-504 (2008).</li>
<li>Fukuda, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Down-regulation+of+protocadherin-%CE%B1%2C+A+isoforms+in+mice+changes+contextual+fear+conditioning+and+spatial+working+memory%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">Down-regulation of protocadherin-α, A isoforms in mice changes contextual fear conditioning and spatial working memory.</a> Eur J Neurosci. , In Press Forthcoming.</li>
<li>Ikeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+YWHAE%2C+a+gene+encoding+14-3-3epsilon%2C+as+a+possible+susceptibility+gene+for+schizophrenia%5BTitle%5D)+AND+%22Behav+Neurosci%22%5BJournal%5D)">Identification of YWHAE, a gene encoding 14-3-3epsilon, as a possible susceptibility gene for schizophrenia.</a> Behav Neurosci. , In Press Forthcoming.</li>
<li>Korte, S. M., De Boer, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+robust+animal+model+of+state+anxiety%3A+fear-potentiated+behaviour+in+the+elevated+plus-maze%5BTitle%5D)+AND+%22Eur+J+Phamacol%22%5BJournal%5D)">A robust animal model of state anxiety: fear-potentiated behaviour in the elevated plus-maze.</a> Eur J Phamacol. 463, 163-175 (2003).</li>
<li>Hogg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+the+validity+and+variability+of+the+elevated+plus-maze+as+an+animal+model+of+anxiety%5BTitle%5D)+AND+%22Pharmacol+Biochem+Behav%22%5BJournal%5D)">A review of the validity and variability of the elevated plus-maze as an animal model of anxiety.</a> Pharmacol Biochem Behav. 54, 21-30 (1996).</li>
<li>Holmes, A., Parmigiani, S., Ferrari, P. F., Palanza, P., Rodgers, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+profile+of+wild+mice+in+the+elevated+plus-maze+test+for+anxiety%5BTitle%5D)+AND+%22Physiol+Behav%22%5BJournal%5D)">Behavioral profile of wild mice in the elevated plus-maze test for anxiety.</a> Physiol Behav. 71, 509-516 (2000).</li>
<li>Tsujimura, A., Matsuki, M., Takao, K., Yamanishi, K., Miyakawa, T., Hashimoto-Gotoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mice+lacking+the+kf-1+gene+exhibit+increased+anxiety-+but+not+despair-like+behavior%5BTitle%5D)+AND+%22Front.+Behav.+Neurosci%22%5BJournal%5D)">Mice lacking the kf-1 gene exhibit increased anxiety- but not despair-like behavior.</a> Front. Behav. Neurosci. 10, In Press (2008).</li>
<li>Nakajima, R., Takao, K., SM, H. uang, Takano, J., Iwata, N., Miyakawa, T., Saido, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+Behavioral+Phenotyping+of+Calpastatin-Knockout+Mice%5BTitle%5D)+AND+%22Molecular+Brain%22%5BJournal%5D)">Comprehensive Behavioral Phenotyping of Calpastatin-Knockout Mice.</a> Molecular Brain. , In Press Forthcoming.</li>
<li>Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditional+calcineurin+knockout+mice+exhibit+multiple+abnormal+behaviors+related+to+schizophrenia%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Conditional calcineurin knockout mice exhibit multiple abnormal behaviors related to schizophrenia.</a> Proc Natl Acad Sci U S A. 100, 8987-8992 (2003).</li>
<li>Factor analyses of large-scale data justify the behavioral test battery strategy to reveal the functional significances of the genes expressed in the brain. Yamasaki, N. The 36th Annual Meeting of the Society for Neuroscience, 2006 October 14–18, Atlanta, Georgia, , Program number 100.15, Poster Number PP32 (1985).</li>
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</ol>

All procedures were approved by the Animal Use and Care Committee of Kyoto University.

Although the mouse genome has been sequenced, the functions of most of the genes are not known. Genetic modification techniques allow for deletion or other manipulation of a specific gene in mice (Austin et al., 2004; Aiba et al., 2007). The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice (Takao and Miyakawa, 2006b; Takao et al., 2007).
The elevated plus maze test is one of the most popular tests of all currently available animal models of anxiety (Rodgers and Dalvi, 1997; Crawley, 2007). This test for anxiety-like behavior has been used for screening and phenotyping transgenic and knockout mice (Crawley, 1999) and for drug discovery (Hogg et al. 1996; Crawley, 2007). The elevated plus maze test has a strong predictive validity for screening anxiolytic drugs (Rodgers and Dalvi, 1997; Mechiel Korte and De Boer, 2003; Crawley, 2007); anxiolytic drugs specifically increase, and anxiogenic drugs specifically decrease, the number of entries into the open arms and the time spent there. The total entries score and total distance are considered a useful index of general activity. Total entries score is also an index of anxiety, and the percentages of entries and time spent in each arm constitute the index of primary anxiety (Rodgers and Dalvi, 1997, Mechiel Korte and De Boer, 2003). The open and closed arms are considered to evoke the same exploratory drive, therefore avoidance of the open arms is considered to be a result of the induction of higher levels of fear (Rodgers and Dalvi, 1997). It is thought that the aversion of mice to explore the open arms of the maze is caused by fear of open and elevated spaces.
In 1984, Handley and Mithani reported on preliminary work with the elevated X (plus) maze test described above. The original test apparatus was raised 70 cm above the floor, and comprised two closed and two open arms, each of which measured 45 cm long by 10 cm wide. In their initial studies, they reported the ratio of open/total arm entries (Handley and Mithani, 1984). Subsequently, other indices were developed that included the number of entries into the closed and open arms and the time spent in the closed and open arms for rats (Pellow et al, 1985; Pellow and File, 1986) and mice (Lister, 1987).
Changes to the elevated plus maze test included lengthening of both the open arms (50 x 10 cm) and closed arms (50 x 10 x 40 cm) with tall surrounding walls and an open roof on the closed arms, and the entire maze was elevated to a height of 50 cm (Pellow et al, 1985; Pellow and File, 1986). Presently, in our laboratory, the elevated plus maze test apparatus is configured in the shape of a +, with two open arms (25 x 5 cm, with a very slight, 0.5-cm, wall) across from each other and perpendicular to two closed arms (25 x 5 x 16 cm), and is raised 50 cm above the floor (Miyakawa et al, 1996; Manabe et al, 2000; Miyakawa et al, 2001; Seeger et al, 2004; Morishima et al, 2005; Miyamoto et al, 2005; Arrow et al, 2006; Hattori et al, 2007; Niemann et al, 2007; Sano et al, 2008; Horii et al, 2008; Fukuda et al, 2008; Ikeda et al; 2008). The mouse is placed in the center of the + (5 cm x 5 cm) and is allowed to explore the maze freely. Though 5 min recording is common, the behavior is recorded for 10 min in our protocol to increase the opportunity to detect the phenotype. The open and closed elevated arms induce an exploration conflict (Mechiel Korte and De Boer, 2003; Crawley, 2007).
The measures of the elevated plus maze test are recorded by an observer during the experiment. In our laboratory, the test is recorded with a video camera connected to a computer and the behavioral data (Image EP) are acquired and analyzed using the Image EP program. Number of entries onto the open arms versus number of total arm entries, and time spent on the open arms versus closed arms, provide the measures of anxiety-like behavior.
We assessed more than 90 strains of genetically engineered mutant mice using the protocol shown in the movie and have a large set of raw data for more than 5000 mice (including wild-type and mutant mice). In our test battery, wild-type littermates are usually used as a control. As a background strain, C57BL/6J mice are widely used. We collected the data of C57BL/6J mice in the behavioral tests. The values obtained from C57BL/6J mice in our elevated plus maze test are as follows (n=914; mean SEM); total distance traveled: 1547.55 14.27 cm; duration of time spent: 56.49 2.42 s (open arms), 384.02 3.13 s (closed arms), 161.90 2.17 s (maze center); ratio of time spent in the open arms: 9.19 0.36%, ratio of time spent in the closed arms: 63.82 0.52%; number of entries: 7.64 0.21 (open arms), 24.32 0.28 (closed arms); % open arm entries: 21.9 0.05, % closed arms entries: 78.1 0.05. C57BL/6J mice spend less time in open arms than in closed arms (p&lt;0.0001, n=914, paired-t test). This indicates that C57BL/6J mice tend to avoid the open arms, and that the time spent in the open arms is a valid index of anxiety-like behavior. Additionally, the order of trials is counterbalanced across genotypes, because the trial number affects the time spent in the open arms and the center platform. That is, the indices all increase during the 3rd and 4th mice compared to the 1st mice (p=0.0089, n=476) (unpublished data). With our protocol, analysis of more than 1661 mice showed that the sequential order of the mice tested in a cage does not significantly affect the open arm stay time (unpublished data). That is, the performances of the first mice taken from the cage do not significantly differ from those of the second, the third or the last mice.
Although the elevated plus maze test and the light/dark transition test are both used for assessing anxiety-like behavior, the results are not always consistent between them (Holmes et al, 2000; Tujimura et al, 2008; Nakajima et al, in press) For example, forebrain-specific calcineurin-knockout mice spend a decreased amount of time in the light chamber in the light/dark transition test, but an increased amount of time in the open arms in the elevated plus maze test (Miyakawa et al., 2003). Factor analysis of our behavioral test battery indicates that the elevated plus maze test and the light/dark transition test assess different aspects of anxiety-like behavior, such as bright-space anxiety in the light/dark transition test and open-space anxiety-like behavior in the open field test (Takao and Miyakawa, 2006b; Yamasaki et al, 2006). Accordingly, both the light/dark transition test and the elevated plus maze test are included in our behavioral test battery.
Crabbe and colleagues reported that uncontrolled variables and experiments characterizing mutants may yield results that are specific to a particular laboratory (Crabbe et al., 1999). The procedural differences that exist between laboratories make it difficult to replicate or compare the results among them. Establishing visual documentation of the protocol will promote better understanding of experimental procedures, allowing for standardization of the protocols used across laboratories and for comparisons of the behavioral phenotypes of various strains of mutant mice assessed using these tests. We previously published a movie protocol of the light/dark transition test (Takao and Miyakawa, 2006a). Likewise, movies of other protocols, such as for the open field test, the porsolt forced swim test, and fear conditioning tests that we use in our behavioral test battery are presently being made for publication as future video journal articles.

<table><tbody><tr><td>Elevated plus maze</td><td>Tool</td><td>O&amp;rsquo;hara Co.</td><td>(none)</td><td></td></tr></tbody></table>

elevated plus maze for mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


Watch this Scientific Journal Video about Elevated Plus Maze for Mice at JoVE.com

Elevated Plus Maze for Mice

The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

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90.5K Views.  Graduate School of Medicine, Kyoto University. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Video: Elevated Plus Maze for Mice

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.  ...

Behavior

Low-stress Route Learning Using the Lashley III Maze in Mice

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other aversive stimuli to motivate task acquisition. As animals age, sensory modalities deteriorate. For example, many strains of mice develop hearing deficits or cataracts. Changes in the sensory systems required to guide mice during task acquisition present potential confounds in interpreting learning changes in aging animals. Moreover, the use of aversive stimuli to motivate animals to learn tasks is potentially confounding when comparing mice with differential sensitivities to stress. To minimize these types of confounding effects, we have implemented a modified version of the Lashley III maze. This maze relies on route learning, whereby mice learn to navigate a maze via repeated exposure under low stress conditions, e.g. dark phase, no food/water deprivation, until they navigate a path from the start location to a pseudo-home cage with 0 or 1 error(s) on two consecutive trials. We classify this as a low-stress behavior test because it does not rely on aversive stimuli to encourage exploration of the maze and learning of the task. The apparatus consists of a modular start box, a 4-arm maze body, and a goal box. At the end of the goal box is a pseudo-home cage that contains bedding similar to that found in the animal&#x2019;s home cage and is specific to each animal for the duration of maze testing. It has been demonstrated previously that this pseudo-home cage provides sufficient reward to motivate mice to learn to navigate the maze1. Here, we present the visualization of the Lashley III maze procedure in the context of evaluating age-related differences in learning and memory in mice along with a comparison of learning behavior in two different background strains of mice. We hope that other investigators interested in evaluating the effects of aging or stress vulnerability in mice will consider this maze an attractive alternative to behavioral tests that involve more stressful learning tasks and/or visual cues.

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other ...

Neuroscience

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 ...

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.

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 ...

Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...

Elevated Plus Maze Test Combined with Video Tracking Software to Investigate the Anxiolytic Effect of Exogenous Ketogenic Supplements

The overall goal of this study is to describe the methodology of the elevated plus maze (EPM) test in combination with a video tracking software. The purpose of the method is to document the effect of various potential anxiolytic treatments on laboratory rodent models. The EPM test is based on the rodents&#39; proclivity toward protected, enclosed dark spaces and unconditioned fear of open spaces and heights, and their innate intense motivation to explore novel environments. The EPM test is a widely used behavioral test for investigating the anxiolytic or anxiogenic responses of rodents given drugs that are known to affect behavior. Observation demonstrating a decreased proportion of time spent on closed arms, an increased proportion of time spent on open arms, a reduced number of entries to closed arms, and an elevated number of entries to open arms measured by the EPM test may reflect reduced anxiety levels. Using this method, the effect of exogenous ketone supplements on anxiety-related behavior is tested in Sprague Dawley (SPD) rats. Exogenous ketone supplements are chronically fed to the rats for 83 days or subchronically and acutely orally gavaged, daily for 7 days, before conducting the EPM test. Behavioral data collection is performed using the SMART video tracking system by a blinded observer at the end of the treatments. The main findings indicate that the EPM test is an effective method to detect the ketone supplement-induced anxiolytic effect and can be considered a sensitive measure to assess changes in anxiety behavior associated with drug- or metabolic-based therapies.

Here, we present a protocol to investigate changes in the anxiety level of rodent animal models. The elevated plus maze (EPM) test, used together with a video tracking software, provides a reliable method to document the effect of various potential anxiolytic treatments in preclinical laboratory scenarios.

The overall goal of this study is to describe the methodology of the elevated plus maze (EPM) test in combination with a video tracking software. The ...

Chronic Stress Shifts Effort-Related Choice Behavior in a Y-Maze Barrier Task in Mice

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that measures motivation to expend effort and obtain reward. In mice, chronic stress exposure significantly impacts motivation to work for a higher value reward when a lesser value reward is freely available compared to unstressed mice. Here we describe the chronic corticosterone administration paradigm, which produces a shift in effortful responding in the Y-maze barrier task. In the Y-maze task, one arm contains 4 food pellets, while the other arm contains only 2 pellets. After mice learn to select the high reward arm, barriers with progressively increasing height are then introduced into the high reward arm over multiple test sessions. Unfortunately, most chronic stress paradigms (including corticosterone and social defeat) were developed in male mice and are less effective in female mice. Therefore, we also discuss chronic non-discriminatory social defeat stress (CNSDS), a stress paradigm we developed that is effective in both male and female mice. Repeating results with multiple distinct chronic stressors in male and female mice combined with increased usage of translationally relevant behavior tasks will help to advance the understanding of how chronic stress can precipitate mood disorders.

The Y-maze barrier task is a behavior test that examines motivation to expend effort for reward. Here, we discuss testing multiple well-validated chronic stressors including chronic corticosterone and social defeat stress with this behavior, as well as the novel chronic non-discriminatory social defeat stress (CNSDS), which is effective in females.

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that ...

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as photophobia. Whilst it is impossible to confirm that a mouse is experiencing migraine, light aversion can be used as a behavioral surrogate for the migraine symptom of photophobia. To test for light aversion, we utilize the light/dark assay to measure the time mice freely choose to spend in either a light or dark environment. The assay has been refined by introducing two critical modifications: pre-exposures to the chamber prior to running the test procedure and adjustable chamber lighting, permitting the use of a range of light intensities from 55 lux to 27,000 lux. Because the choice to spend more time in the dark is also indicative of anxiety, we also utilize a light-independent anxiety test, the open field assay, to distinguish anxiety from light-aversive behavior. Here, we describe a modified test paradigm for the light/dark and open field assays. The application of these assays is described for intraperitoneal injection of calcitonin gene-related peptide (CGRP) in two mouse strains and for optogenetic brain stimulation studies.

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as ...

Validating a Murine Model of Angelman Syndrome Using Behavioral Tests

Behavioral Characterization of an Angelman Syndrome Mouse Model

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of AS. We use the rotarod learning paradigm, detailed gait analysis, and nest building test to detect and characterize animal motor impairments. We test animal emotionality in the open field and elevated plus maze tests, as well as the affect in the tail suspension test. When AS mice are tested in the open field test, the results should be interpreted with care, since motor dysfunctions influence mouse behavior in the maze and alter activity scores.
The reproducibility and effectiveness of the presented behavioral tests has already been validated in several independent Uba3a mouse lines with different knockout variants, establishing this set of tests as an excellent validation tool in AS research. Models with the relevant construct and face validity will warrant further investigations to elucidate the pathophysiology of the disease and grant the development of causal treatments.

Author Spotlight: A Battery of Highly Reproducible Behavioral Tests to Validate an Angelman Syndrome Murine Model 

This manuscript presents a set of highly reproducible behavioral tests to validate an Angelman syndrome mouse model.

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of ...