Barnes Maze Testing Strategies with Small and Large Rodent Models

Summary

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.

Abstract

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. 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. distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e. 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. 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.

Introduction

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. 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. degus [Octodon degus])^8-13. Other species assessed using the Barnes maze include American cockroaches (Periplaneta americana)¹⁴, corn snakes (Elaphe guttata guttata)¹⁵, squamate reptiles (e.g. side-blotched lizards [Uta stansburiana])¹⁶, and nonhuman primates (e.g. 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) exposure^9-1113. It is also commonly used for behavior phenotyping of various mouse strains^18-21, assessment of aging effects^7,22-28, and Alzheimer's Disease-related deficits in animal models^3,29-33, as well as the effects of exercise and dietary, environmental, and metabolic alterations^34-42.

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 mice^3,46,47, Barnes maze performance is more sensitive to certain other alterations^48,49. 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. 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's spatial navigational learning and memory ability^8-10. 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'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 "patterns" 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 species^18-21.

Protocol

1. Barnes Maze Procedure for Small Rodents

1. Turn on the overhead lights above the maze and place "Do Not Enter" signs on the outside of the lab door.
2. 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.
3. Set up the tracking program.
4. 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.
   1. Ensure the paper that is blocking the tube is removed from that escape hole and all other holes are plugged.
   2. Draw curtain around the maze.
5. 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.
   1. After placing the animal in the center of the maze, quietly move to the computer area (~150 cm from the maze).
   2. 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.
6. Observe animal'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.
   1. Search strategy is categorized as Direct (going directly into the escape cage with 3 or fewer errors),
   2. Serial (traveling along the maze perimeter until the escape cage is located), or
   3. Random (crossing the maze center multiple times to check various holes).
7. Stop the tracking program when the animal has all four paws inside the escape cage.
8. 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.
9. Remove mouse from the escape cage and place in home cage.
10. Spray the maze top and escape cage with 70% ethanol and wipe dry. Set the first cage/mouse aside for 30 min before running its second trial.
11. 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.
12. Each mouse is tested for 2 trials/day with an inter-trial interval of approximately 30 min.
13. 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.

2. Barnes Maze Testing for Rats When a Tracking Program is Not Available

1. 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.
2. Ensure computer and camera are ready and a stopwatch is available.
3. Turn on white noise to attenuate any noises from other nearby locations. The tester's chair is approximately 122 cm from nearest edge of the maze top and remains in the same location throughout testing.
4. A timer (set to 2 min) should be available (only needed on Day 1 of testing). Timer should not "beep" or otherwise make noise. Door(s) to test room should have "Do Not Enter" sign on outside.
5. 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).
6. 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.
7. 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.

Initial Testing Day 1:

1. 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.
2. 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.
3. 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.
4. 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's chair.
5. 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.
6. 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.
7. 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.
   1. This 15 sec duration can be timed using a clock with a second hand on the test room wall.
   2. Record time of day on the test order sheet and record that the rat did not find the escape cage.
8. 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.
   1. 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.
   2. 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).
   3. Data from trials in which an animal fell/jumped and could not be retrieved within 10 sec are omitted from statistical analyses.
9. Stop video recording on the computer. Record any comments about the trial.
10. 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.
11. 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).
12. Set the center tube with ID sheet for the next subject in the maze center. Begin video recording on the computer.
13. 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.
14. After all animals are tested, clean the maze and escape cage, turn off overhead lights, and white noise. Remove "Do Not Enter" sign(s) from door(s).

Days 2 through 7 Testing

1. Set up test room and maze for testing as detailed above.
2. 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.
3. 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.
4. Repeat procedure beginning from step 4 above.

3. Statistical Analyses for Barnes Maze Endpoints

1. 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.
2. 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.
   1. This statistical method is useful for behavioral data in which there is an upper limit cutoff.
3. Search strategy data may be analyzed by using a repeated measure design with PROC GLIMMIX and SAS version 9.2 software analysis.
   1. 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.
   2. 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.
   3. This latter method results in a binomial distribution and also employs PROC GLIMMIX.

Results

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 ...

Discussion

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. 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...

Materials

Name	Company	Catalog Number	Comments
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.
Barnes Maze platform with 12 or 20 escape holes every 30°. 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.	US Plastics Corp, Lima, OH	42625	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.
2 in Polypropylene pipe plug (24) 2 in 90° Black polypropylene elbow (12) 2 in x 6 in Polypropylene pipe nipple (1)	US Plastics Corp, Lima, OH	30724 32086 30712	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.
False bottoms for rat Barnes Maze			These were custom made of ABS plastic and vacuum molded for the rat maze apparatus.
Circular aluminum wall/barrier (50 cm high) around the maze	Ace Hardware, Columbia, MO		In the case of small rodents (e.g., 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.
Support stand for maze platform top	US Plastics Corp, Lima, OH	42625	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.
White noise	SleepMate Sound Conditioner, Marpac, Rocky Point, NC	980A	Background noise may be used to block out peripheral acoustic cues that may confound Barnes Maze testing across trials and animals
Light fixtures and 300-500 W bulbs encased in aluminum shells. For example, Utilitech 500 W halogen portable work lights.	Ace Hardware or Lowes		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.
Escape cage. For small rodents, this can be a polypropylene cage (27.8 cm x 7.5 cm x 13 cm).	Ancare, Bellmore, NY	N40 PP	The rat escape cage here was custom built and has a ramp leading into the escape cage.
Opaque tube (rats only) (27 cm diameter; 23 cm height) with a piece of thick cardboard to cover the top.			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.
High resolution video camera (e.g., Panasonic Digital Video Camera)	Panasonic, Secaucus, NJ	ICV19458	The video camera is positioned overhead and records trials for later analysis.
Extra- or intra-maze geometric cues made of high quality cardboard construction paper	any office supply store, such as Staples		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.
Black curtain to surround the maze (small rodents only)	any fabric and crafts store, such as Jo-Ann Fabrics		A black curtain is used in small rodents (especially wild species, e.g. Peromyscus) to maintain attention within the maze confines.
70% Ethanol	Fisher Scientific	BP2818-4	After each trial, the maze top and escape cage are cleaned to eliminate potential odor cues for consecutively tested rodents.
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.).	Noldus (Leesburg, VA)		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.
External hard drives, such as Seagate or WD, with a minimum 1-2 TB of memory	Any office supply store, such as Staples.		Videorecordings should be backed up in at least one separate location.
Videorecording program, e.g. WinTV program	Hauppauge Computer Works, Inc., Hauppauge, NY		If tracking software is not available at the time of the testing, the trials should be video-recorded for later analysis