The authors acknowledge Mr. Eldin Ja&#353;arevi&#263;, Mr. Scott Williams, Mr. Roger W. Meissen, Sarah A. Johnson,&#160;Dr. R. Michael Roberts, Dr. Mark R. Ellersieck, and Dr. David C. Geary at the University of Missouri, and Mr. C. Delbert Law and the animal care staff at the National Center for Toxicological Research/FDA. This work was supported by an NIH Challenge Grant to Grant to CSR (RC1 ES018195), a Mizzou Advantage Grant to (CSR and DCG), a University of Missouri College of Veterinary Medicine faculty award (CSR), and protocol E7318 at the National Center for Toxicological Research/FDA.

1. Barnes Maze Procedure for Small Rodents
<ol>
	<li>Turn on the overhead lights above the maze and place &#34;Do Not Enter&#34; signs on the outside of the lab door.</li>
	<li>Bring mice in their normal home cages to the test room approximately 30 min prior to beginning the first trial to permit habituation. If the room is quiet, it may not be necessary to include white noise, otherwise this precautionary measure may be considered.</li>
	<li>Set up the tracking program.</li>
	<li>Gently remove the first mouse from its...

Critical steps in Barnes maze testing procedures include: 1) providing the proper mildly aversive stimulus to motivate the animal to locate the escape cage, 2) ensuring uniform conditions are maintained across the animal trials (e.g.&#160;test time, testing personnel, external noise control, and other stimuli that might affect performance), 3) if trials are video recorded, optimizing and ensuring proper video recording and file back up, and 4) cleaning of the maze with 70% ethanol to remove olfactory cues betwee...

Spatial learning and memory in laboratory rodents was first assessed with food-deprived rats that navigated a maze of alleyways to locate a food reinforcer1. Several decades later, a spatial reference memory system was proposed2. In contrast to working memory which refers to memory within a test session or trial, reference memory refers to memory across test sessions or trials and is more closely related to long-term memory.
Several types of mazes have been developed as noninvasive assessments of this hippocampal-dependent spatial learning and memory in small and large rodents (e.g.&#160;water maze, multiple T-maze, radial arm maze and dry-land mazes)3-6. Here, we focus on the circular platform or Barnes maze, first described in 1979 by Dr. Carol Barnes7. This maze has been used to test spatial navigational learning and memory in a wide range of rodent models, including rats (Rattus norvegicus), mice (Mus musculus), deer mice (Peromyscus maniculatus bairdii), California mice (Peromyscus californicus), and hystricomorph rodents (e.g.&#160;degus [Octodon degus])8-13. Other species assessed using the Barnes maze include American cockroaches (Periplaneta americana)14, corn snakes (Elaphe guttata guttata)15,&#160;squamate reptiles (e.g.&#160;side-blotched lizards [Uta stansburiana])16, and nonhuman primates (e.g.&#160;mouse lemurs [Microcebus murinus])17. In our labs, Barnes maze performance has been used as an index of neurotoxicity after developmental bisphenol A (BPA) or ethinyl estradiol (EE2) exposure9-1113. It is also commonly used for behavior phenotyping of various mouse strains18-21, assessment of aging effects7,22-28, and Alzheimer&#39;s Disease-related deficits in animal models3,29-33,&#160;as well as the effects of exercise and dietary, environmental, and metabolic alterations34-42.
A primary advantage of Barnes maze use is that it induces less stress in the subjects relative to water mazes, such as the Morris water maze43, although both can induce acute increases in plasma corticosterone concentrations in mice44. As a dry land maze, the Barnes maze may be more ethologically-relevant for terrestrial rodents45. Although water maze performance has been shown to be more sensitive to genetic alterations in mice3,46,47, Barnes maze performance is more sensitive to certain other alterations48,49. In rodent models where water maze use is not possible, the Barnes maze may provide a fine-tuned assessment of spatial memory retention31. The mildly aversive stimuli typically used in the Barnes maze (i.e.&#160;bright lights), however, may not provide sufficient motivation for the rodent to locate the escape cage45. Furthermore, rodents can learn that no punishment occurs if they do not enter the escape cage. Thus, instead of actively searching for the escape cage, some rodents actively explore the maze for long durations of each trial. As reviewed by Kennard and Woodruff-Pak24, this increased exploration will prolong the latency to locate the escape cage, path length, and increase the number of errors. Thus, measurement of multiple parameters, including latency, error rate, time spent in the correct and incorrect quadrants, velocity, time moving, time resting, and search strategy, may collectively provide a better indicator of each subject&#39;s spatial navigational learning and memory ability8-10. Additionally, performance can be measured as the latency to first locate the escape cage (primary measure) or the latency to enter the escape cage (total measure). Some have argued that primary measures of performance are a more accurate reflection of spatial learning than total measures50. Most studies, including the examples described here, use latency to enter the escape cage to determine error rate and search strategy. Further, some tracking software systems have a three point body detection system that can measure the frequencies of sniffing the correct vs. incorrect holes. Finally, the maze must be thoroughly cleaned with ethanol between trials to remove olfactory cues that could provide cues or prove distracting to subsequent animals.
Barnes maze designs vary but generally each has 12 or 20 potential escape holes, only one of which leads to the home or an escape cage. The escape cage may be situated either directly below the escape hole on the maze top (for mazes without walls) or built into the surrounding wall of the maze. The cues can vary in size from approximately 16.5 cm height or width (within the maze) to a horizontal line 21.6 cm in width placed from floor to ceiling of the room wall outside the maze. Figures 1-5 show examples of Barnes maze designs for Peromyscus species (Figure 1) and rats (Figures 2-5). Plugs or false bottoms must cover the nonescape holes to prevent the animal from falling out of the maze. Size of the test room can vary (~20 m2) but it must be large enough to provide ample room for the maze, habituating the animals to the room, accommodating a computer with video set-up (if used), and a place for the experimenter to sit at a distance (at least ~122 cm) from the maze apparatus such that their presence does not interfere with the animal&#39;s performance. Assignment of escape cage location should be balanced among treatment groups and sex. While the specific procedures described here do not include rotating the maze between trials to discourage use of the intra-maze odor cues, some studies incorporate this procedure50. In our procedures, the maze is wiped clean with ethanol between trials to eliminate odor cues.
In locating the escape cage, three types of search strategies have been defined (originally termed &#34;patterns&#34; by Barnes7): 1) random, operationally defined as localized searches of holes separated by paths crossing the maze center, 2) serial, defined as a systematic search of consecutive holes in a clockwise or counterclockwise direction, and 3) direct or spatial, defined as navigating directly to the correct quadrant without crossing the maze center more than once and with three or fewer errors. In general, with repeated testing, rodents typically progress through the search strategies in the order listed (random, serial, and direct)51. A probe trial without the escape cage may also be used as a further measure of memory50.
The protocol and representative results here were developed for two types of rodents (Peromyscus species- otherwise termed small rodents) and rats. While these general procedures may also hold for inbred and/or outbred mice (Mus musculus), other studies should be consulted on potential methodology differences for those latter species18-21.

<table border="0" cellpadding="0" cellspacing="0" width="1285">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:22px;width:1287px;">NOTE: Those items that are for small rodents only are bolded. Those items that are for large rodents only are italicized. Items neither bolded nor italicized are for both.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:335px;">Barnes Maze platform with 12 or 20 escape holes every 30&#176;. For rats, each hole is 10.5 cm in diameter and 4 cm from the maze top edge. For use with automated tracking programs, a black top for white rodents or a white top for pigmented rodents is needed. For mice and rats, this circular top is 95 and 122 cm in diameter, respectively.</td>
			<td>US Plastics Corp, Lima, OH</td>
			<td>42625</td>
			<td style="width:469px;">This is the top of the Barnes Maze and the surface that the rodent is placed upon. It can be constructed from a variety of materials (e.g. Plexiglas), but for endocrine disruptor work, polypropylene BPA-free material is optimal. One of the holes leads to the an escape cage; all other holes are blind-ending or false-bottomed. For the rat maze, small slides on the underside of the maze platform allow the escape cage and false bottoms to slide in.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:335px;">2&#34; Polypropylene pipe plug (24) 
			2&#34; 90&#176; Black polypropylene elbow (12) 
			2&#34; x 6&#34; Polypropylene pipe nipple (1)</td>
			<td>US Plastics Corp, Lima, OH</td>
			<td style="width:215px;">30724 
			32086 
			30712</td>
			<td style="width:469px;">These are only necessary for the small rodent (e.g. mouse) Barnes Maze. These adaptations are either blind-ending tubes/elbows or one of the tubes is connected to the pipe nipple that then leads to the escape cage.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:335px;">False bottoms for rat Barnes Maze</td>
			<td></td>
			<td style="width:215px;"></td>
			<td style="width:469px;">These were custom made of ABS plastic and vacuum molded for the rat maze apparatus.</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:335px;">Circular aluminum wall/barrier (50 cm high) around the maze</td>
			<td style="width:268px;">Ace Hardware, Columbia, MO</td>
			<td style="width:215px;"></td>
			<td style="width:469px;">In the case of small rodents (e.g.&#160;mice), this barrier prevents them from falling off the maze; the rat apparatus generally does not require this. The wall may not be needed for laboratory mice that are relatively tame.</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:335px;">Support stand for maze platform top</td>
			<td style="width:268px;">US Plastics Corp, Lima, OH</td>
			<td style="width:215px;">42625</td>
			<td style="width:469px;">The stand supports the maze platform top such that it is elevated above the floor (typically, 70-100 cm) to motivate the rodent to locate the escape cage. The stand can be constructed of any material.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:65px;width:335px;">White noise</td>
			<td style="width:268px;">SleepMate Sound Conditioner, 
			Marpac, Rocky Point, NC</td>
			<td style="width:215px;">980A</td>
			<td style="width:469px;">Background noise may be used to block out peripheral acoustic cues that may confound Barnes Maze testing across trials and animals</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:86px;width:335px;">Light fixtures and 300-500 watt bulbs encased in aluminum shells. For example, Utilitech 500-watt halogen portable work lights.</td>
			<td style="width:268px;">Ace Hardware or Lowes</td>
			<td style="width:215px;"></td>
			<td style="width:469px;">Bright lights provide a mildly aversive stimulus which motivate the rodent to locate the escape cage. The lights are generally suspended ~150 cm above the maze top.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Escape cage. For small rodents, this can be a polypropylene cage (27.8 x 7.5 x 13 cm).</td>
			<td style="width:268px;">Ancare, Bellmore, NY</td>
			<td style="width:215px;">N40 PP</td>
			<td style="width:469px;">The rat escape cage here was custom built and has a ramp leading into the escape cage.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:335px;">Opaque tube (rats only) (27 cm diameter; 23 cm height) with a piece of thick cardboard to cover the top.</td>
			<td style="width:268px;"></td>
			<td style="width:215px;"></td>
			<td style="width:469px;">The tube is placed in the center of the maze and the rat is placed into the tube from the top which is covered with the cardboard. A handle on the outside of the tube allows easier lifting of the tube, which then begins the trial. The tube can be constructed of any material, but should be opaque.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:62px;width:335px;">High resolution video camera (e.g.&#160;Panasonic Digital Video Camera)</td>
			<td style="width:268px;">Panasonic, Secaucus, NJ</td>
			<td style="width:215px;">ICV19458</td>
			<td style="width:469px;">The video camera is positioned overhead and records trials for later analysis.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:335px;">Extra- or intra-maze geometric cues made of high quality cardboard construction paper</td>
			<td style="width:268px;">Any office supply store, such as Staples.</td>
			<td style="width:215px;"></td>
			<td style="width:469px;">These visual cues orient the animal within the maze environment, providing cues as to the spatial location of the escape cage; in rats, extra-maze cues on the walls work well, whereas in small rodents that require a wall around the maze, intra-maze cues must be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Black curtain to surround the maze (small rodents only)</td>
			<td style="width:268px;">Any fabric and crafts store, such as Jo-Ann Fabrics</td>
			<td style="width:215px;"></td>
			<td style="width:469px;">A black curtain is used in small rodents (especially wild species, e.g. Peromyscus) to maintain attention within the maze confines.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">70% ethanol</td>
			<td style="width:268px;">Fisher Scientific</td>
			<td>BP2818-4</td>
			<td style="width:469px;">After each trial, the maze top and escape cage are cleaned to eliminate potential odor cues for consecutively tested rodents.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:335px;">Tracking software program, such as Ethovision, and computer with appropriate video card and substantial (1 TB or more) hard-drive space. Alternatively, videos can be recorded directly to the computer for later analysis using a program such as Win TV (Hauppauge Computer Works, Inc.).</td>
			<td style="width:268px;">Noldus (Leesburg, VA)</td>
			<td style="width:215px;"></td>
			<td style="width:469px;">Tracking software is required to analyze trials for latency to locate the escape cage, velocity, distance traveled, time spent resting, time spent moving, time spent in the correct versus incorrect quadrants, time spent around the escape hole, number of errors or entries into incorrect holes, and overall search strategy employed to find the escape cage.</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:87px;width:335px;">External hard drives, such as Seagate or WD, with a minimum 1- 2 TB of memory</td>
			<td>Any office supply store, such as Staples.</td>
			<td></td>
			<td>Videorecordings should be backed up in at least one separate location.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Videorecording program, e.g. WinTV program</td>
			<td style="width:268px;">Hauppauge Computer Works, Inc., 
			Hauppauge, NY</td>
			<td></td>
			<td style="width:469px;">If tracking software is not available at the time of the testing, 
			the trials should be video-recorded for later analysis</td>
		</tr>
	</tbody>
</table>

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.

Sexually mature male deer mice are dependent upon enhanced spatial navigational ability to locate potential breeding partners, which are widely disseminated throughout the environment. Both prenatal and adult exposure to testosterone are essential in organizing and activating this later adult male behavior53. As such, it was presumed that early exposure to endocrine disrupting compounds might disturb this later trait in males. To test this hypothesis, male and female deer mice were developmentally exposed ...

Watch this Scientific Journal Video about Barnes Maze Testing Strategies with Small and Large Rodent Models at JoVE.com

Barnes Maze Testing Strategies with Small and Large Rodent Models

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 ...

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41.0K Views.  University of Missouri. 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. 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.