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 home cage and place in the tall covered plastic box. Place its escape (clean home) polypropylene cage (29 cm x 19 cm x 13 cm) under the designated escape hole.
	<ol>
		<li>Ensure the paper that is blocking the tube is removed from that escape hole and all other holes are plugged.</li>
		<li>Draw curtain around the maze.</li>
	</ol>
	</li>
	<li>Place the plastic box with the mouse inside in the maze center and approximately 8 sec later, gently take the animal out of the cage and place it onto the maze.
	<ol>
		<li>After placing the animal in the center of the maze, quietly move to the computer area (~150 cm from the maze).</li>
		<li>Initiate the appropriate tracking software program that should already be open to ensure minimal time (within a few seconds) has elapsed from the time the animal was placed on the maze until the program starts documenting its performance.</li>
	</ol>
	</li>
	<li>Observe animal&#39;s performance from computer monitor and record hole number, trial number, search strategy, and number of errors made. An error is defined as sniffing of an incorrect hole. Assessment of search strategy may be made live or later based on the tracking pattern.
	<ol>
		<li>Search strategy is categorized as Direct (going directly into the escape cage with 3 or fewer errors),</li>
		<li>Serial (traveling along the maze perimeter until the escape cage is located), or</li>
		<li>Random (crossing the maze center multiple times to check various holes).</li>
	</ol>
	</li>
	<li>Stop the tracking program when the animal has all four paws inside the escape cage.</li>
	<li>If the mouse fails to enter the escape cage within 5 min, gently guide it to the correct location and into the escape cage. Let the mouse remain in the escape cage for 2 min.</li>
	<li>Remove mouse from the escape cage and place in home cage.</li>
	<li>Spray the maze top and escape cage&#160;with 70% ethanol and wipe dry. Set the first cage/mouse aside for 30 min before running its second trial.</li>
	<li>Before beginning the next mouse, plug the previously correct escape hole and remove the paper plug blocking the hole from the designated escape hole for that next subject.</li>
	<li>Each mouse is tested for 2 trials/day with an inter-trial interval of approximately 30 min.</li>
	<li>Repeat these steps until all mice have been tested for seven consecutive days, which may increase the likelihood of observing improved performance and/or differences between treatment groups, relative to only four days of data.</li>
</ol>
2. Barnes Maze Testing for Rats When a Tracking Program is Not Available
<ol>
	<li>Ensure the maze is in its correct placement (directly centered below lights), false bottoms that block nonescape holes and prevent the animal from falling out are securely in the maze, and the escape cage is in the designated location for the first subject. Overhead lights above the maze should be turned on.</li>
	<li>Ensure computer and camera are ready and a stopwatch is available.</li>
	<li>Turn on white noise to attenuate any noises from other nearby locations. The tester&#39;s chair is approximately 122 cm from nearest edge of the maze top and remains in the same location throughout testing.</li>
	<li>A timer (set to 2 min) should be available (only needed on Day 1 of testing). Timer should not &#34;beep&#34; or otherwise make noise. Door(s) to test room should have &#34;Do Not Enter&#34; sign on outside.</li>
	<li>A test order sheet for the subjects will list the order of subject testing, the session number, the hole number location of the escape cage for each subject, and areas to record latency and time of day for each subject as well as an area for any necessary notes (Figure 6).</li>
	<li>From 30-60 min before the first rat is to be tested, bring the animals in their home cages to the test room to allow for habituation.</li>
	<li>The center tube that the rat is placed into at the beginning of a trial is set in the maze center. Set the cardboard sheet showing the first animal ID on top. This allows the video recording to capture the animal ID for easy identification of each subject by observing the first few seconds of the video.</li>
</ol>
Initial Testing Day 1:
<ol>
	<li>Begin computer video recording (if used) and include approximately 5 sec of the trial with animal ID sheet for subject identification. File name (or date created) will identify day/time of testing.</li>
	<li>Remove the first animal from its home cage (check identity if multiple animals are in cage) and gently place head first into the escape cage. Cover the escape cage with an extra false bottom and start the 2 min timer. This allows the animal to habituate to the escape cage.</li>
	<li>After 2 min timer ends, gently remove animal from escape cage (remove false bottom cover as well and set away from maze), lift ID sheet, and immediately place the rat inside the center tube. Cover top of center tube with cardboard ID sheet.</li>
	<li>Gently and slowly lift center tube with cardboard cover and set aside. Start the stopwatch as the center tube is lifted above the animal. Move to sit in the tester&#39;s chair.</li>
	<li>Sit quietly in the chair, watching both the animal and the stopwatch. Each animal has a maximum of 5 min to find the escape cage.</li>
	<li>If the rat finds the escape cage in less than 5 min, stop the stopwatch and record latency and time of day on test order sheet. Remove animal from escape cage and place back into home cage.</li>
	<li>If the rat does not find the escape cage within 5 min, gently guide the animal to the escape cage and allow 15 sec to pass before removing and returning the animal to the home cage.
	<ol>
		<li>This 15 sec duration can be timed using a clock with a second hand on the test room wall.</li>
		<li>Record time of day on the test order sheet and record that the rat did not find the escape cage.</li>
	</ol>
	</li>
	<li>If the rat falls/jumps off the maze, the tester should glance at stopwatch for time. The tester should then attempt to quickly retrieve the animal.
	<ol>
		<li>If this can be done within 10 sec, replace the animal onto the center of the maze and record the time of the fall/jump on the test sheet (if the tester can distinguish between a fall or jump, this should be denoted). Continue the trial.</li>
		<li>If retrieving the animal takes longer than 10 sec, stop the stopwatch, and put animal back into the home cage. Record time of fall/jump (if the tester can distinguish between a fall or jump, note this).</li>
		<li>Data from trials in which an animal fell/jumped and could not be retrieved within 10 sec are omitted from statistical analyses.</li>
	</ol>
	</li>
	<li>Stop video recording on the computer. Record any comments about the trial.</li>
	<li>Remove any urine or feces from the maze top, spray with 70% ethanol, and thoroughly wipe dry. Remove escape cage and clean with 70% ethanol.</li>
	<li>Put a clean escape cage at the designated placement for the next subject. Having more than one escape cage allows each to air-dry to lessen the ethanol odor. Put a clean false bottom at the previous hole (so that all but one hole has a false bottom and that one hole contains the escape cage).</li>
	<li>Set the center tube with ID sheet for the next subject in the maze center. Begin video recording on the computer.</li>
	<li>Remove the next animal to be tested, place into escape cage (if Day/Session 1), and start 2 min timer (only if Day/Session 1). Continue from step 2 above. Each subject receives 1 trial/day.</li>
	<li>After all animals are tested, clean the maze and escape cage, turn off overhead lights, and white noise. Remove &#34;Do Not Enter&#34; sign(s) from door(s).</li>
</ol>
Days 2 through 7 Testing
<ol>
	<li>Set up test room and maze for testing as detailed above.</li>
	<li>Set center tube in maze center with ID sheet on top. Begin video recording. Remove the first animal from home cage and place into the center tube.</li>
	<li>This step differentiates Days 2-7 from Day 1; specifically, on Days 2-7, the subject is placed directly into the center tube after removal from the home cage and the 2 min habituation period inside the escape cage is not done.</li>
	<li>Repeat procedure beginning from step 4 above.</li>
</ol>
3. Statistical Analyses for Barnes Maze Endpoints
<ol>
	<li>Data analyses may require several statistical tests. Continuous variables, such as latency and error rate, may be analyzed as a split plot in space and time52.</li>
	<li>If some animals do not locate the escape or home cage within the maximum allotted time, the latency data can be assigned as the maximum and analyzed by using ProcLife testing in SAS version 9.2 software analysis.
	<ol>
		<li>This statistical method is useful for behavioral data in which there is an upper limit cutoff.</li>
	</ol>
	</li>
	<li>Search strategy data may be analyzed by using a repeated measure design with PROC GLIMMIX and SAS version 9.2 software analysis.
	<ol>
		<li>This first analysis employs a cumulative log it link and a multinomial distribution such that all three search strategies (random, serial, and direct) are included in this analysis.</li>
		<li>To determine if the animals are learning to use the more efficient search strategy (direct), a second analysis on search strategy can be performed on which the two less efficient strategies (random and serial) are combined and compared against the more efficient direct search strategy.</li>
		<li>This latter method results in a binomial distribution and also employs PROC GLIMMIX.</li>
	</ol>
	</li>
</ol>

This document has been reviewed in accordance with United States Food and Drug Administration (FDA) policy and approved for publication. Approval does not signify that the contents necessarily reflect the position or opinions of the FDA nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the FDA. The authors have no competing interests and nothing to disclose.

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 ...

barnes-maze-testing-strategies-with-small-and-large-rodent-models

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

41.5K 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.

Video: Barnes Maze Testing Strategies with Small and Large Rodent Models

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of ...

Testing for Odor Discrimination and Habituation in Mice

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal&#8217;s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. 
In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.

This manuscript describes a protocol to examine the olfactory system of rodents. The olfactory habituation/dishabituation test will allow investigators to determine whether a mouse habituates to a repeatedly presented odor and whether the mouse demonstrates dishabituation when presented a novel odor.

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the ...

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 ...

Testing Sensory and Multisensory Function in Children with Autism Spectrum Disorder

In addition to impairments in social communication and the presence of restricted interests and repetitive behaviors, deficits in sensory processing are now recognized as a core symptom in autism spectrum disorder (ASD). Our ability to perceive and interact with the external world is rooted in sensory processing. For example, listening to a conversation entails processing the auditory cues coming from the speaker (speech content, prosody, syntax) as well as the associated visual information (facial expressions, gestures). Collectively, the &#8220;integration&#8221; of these multisensory (i.e., combined audiovisual) pieces of information results in better comprehension. Such multisensory integration has been shown to be strongly dependent upon the temporal relationship of the paired stimuli. Thus, stimuli that occur in close temporal proximity are highly likely to result in behavioral and perceptual benefits &#8211; gains believed to be reflective of the perceptual system&#39;s judgment of the likelihood that these two stimuli came from the same source. Changes in this temporal integration are expected to strongly alter perceptual processes, and are likely to diminish the ability to accurately perceive and interact with our world. Here, a battery of tasks designed to characterize various aspects of sensory and multisensory temporal processing in children with ASD is described. In addition to its utility in autism, this battery has great potential for characterizing changes in sensory function in other clinical populations, as well as being used to examine changes in these processes across the lifespan.

We describe how to implement a battery of behavioral tasks to examine the processing and integration of sensory stimuli in children with ASD. The goal is to characterize individual differences in temporal processing of simple auditory and visual stimuli and relate these to higher order perceptual skills like speech perception.

In addition to impairments in social communication and the presence of restricted interests and repetitive behaviors, deficits in sensory processing are ...

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, ...

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

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 ...

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. ...