Place and Response Learning in the Open-field Tower Maze

Summary

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.

Abstract

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 “stress” 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.

Introduction

The open-field tower maze (OFTM) can be used to examine the behavioral and neurobiological mechanisms of spatial learning in rodents. Importantly, the OFTM allows researchers to study both “place-based” and “response-based” learning types. During place-learning an animal navigates through a maze using the spatial cues in the surrounding environment (e.g., a food reward is located next to a tall beacon). During response-learning the animal makes a specific turn response regardless of its position relative to spatial cues (e.g., make a left or right turn to find a food reward). It has been previously reported that response-learning and place-learning rely on two different memory systems^{3, 4} and mapping their neural mechanisms has been an ongoing scientific pursuit^{2, 5-7}. The design of the OFTM was adapted from the one previously described⁸.

Open field paradigms (a component of the OFTM) are commonly used to investigate anxiety in rodents⁹. During the typical open field anxiety test rats that spend more time in the center of an open field have lower stress and anxiety levels than rats that spend more time near the perimeter. In the OFTM, a rat must enter the center of the maze to find the food reward. Therefore, correct navigation in the OFTM indicates that these rats are non-stressed. One reason why the rats are non-stressed during the training is because unlike standard open-field tests of anxiety training the OFTM requires pre-training phase during which the rat is pre-exposed to the maze over a long period of time. In addition, the training occurs in a dimly lit environment further reducing the anxiety-inducing aspect of an open field which is typically brightly lit.

Another unique feature of the OFTM, compared to some other mazes, is how learning is measured. Experimenters using the Morris Water Maze or Barnes Maze typically measure learning by analyzing the speed and/or path length a rat travels to reach its target location. These dependent measures can be affected by non-learning factors, such as changes in a rat’s general motor activity. However, the OFTM measures learning using the percent of first-choice correct response a rat makes to find the food reward. This dependent measure is not vulnerable to changes in motor performance and therefore, more accurately assesses learning than measurements of speed and distance. Nevertheless, the experimenter can also measure a rat’s speed and distance traveled in the OFTM using video tracking software.

Other maze-types, such as the T-maze, Morris Water Maze and Barnes Maze can be used to study place- and response-learning. However, the OFTM is unique in a few notable ways. First, the OFTM is a more sensitive behavioral assay than the T-maze because the OFTM has a chance performance of 25%, whereas the T-maze has a chance performance of 50%. Second, a rat can only turn in one of two ways in the T-maze, but has unrestricted route possibilities when navigating in the OFTM. Third, unlike the Morris Water Maze and Barnes Maze tasks (which have unrestricted route possibilities like the OFTM), experiments using the OFTM do not require rats to navigate through an aversive situation. Rats are motived to navigate in the Morris Water Maze to escape swimming in water and they are motivated to navigate in the Barnes Maze to avoid being in an open, brightly-lit environment. It has been demonstrated that exposure to the Morris Water Maze or Barnes Maze induces the release of stress hormone (i.e., corticosterone) in rodents¹⁰. However, rats navigate in the OFTM because they are motivated to find a food reward and training is conducted with dim ambient lighting. Thus, the OFTM is a non-aversive appetitive task. Results from experiments that use a stressful maze paradigm to examine the effects of manipulations such as drug, hormone, exercise, or diet on learning and memory mechanisms can be difficult to interpret because “stress”, by itself, may influence cognitive function¹¹.

Protocol

This protocol is approved by the Institutional Animal Care and Use Committee of Christopher Newport University.

1. Construction of the OFTM Apparatus

1. Construct the boundary wall (40 cm high and 183 cm diameter) from three pieces of white laminate. Construct the circular maze floor (183 cm diameter) from black laminate and glue it to a plastic table top. Secure the boundary wall to the edge of the table with screws and glue.
2. Decorate the interior surface of the boundary wall: Leave one quarter solid white; Paint one quarter solid black; Paint one quarter with black stripes; Paint one quarter with black circles.
3. Build four “food towers” (8 cm x 8 cm x 20 cm; Figure 1A) cut from a square-shaped white PVC post. Glue a piece of black laminate (8 cm x 8 cm) to the top of each tower. Secure them to the maze floor with crazy glue 54 cm apart from each other.
4. Build three “pre-training towers” (5 cm, 10 cm & 15 cm high respectively; Figure 1B) from a square-shaped white PVC post. Glue a piece of black laminate (8 cm x 8 cm) to the top of each tower. These towers are taped to the maze floor only during the pre-training phase (described below).
5. Glue one black opaque plastic food cup with a screw-top lid (= a clear plastic bead container painted black; 1.75 cm high and 3.75 cm diameter) to the top of each food tower. Drill three small venting holes (3 mm diameter) in each lid. The holes ensure that the rat cannot locate the “correct” food tower using olfactory cues. The height of the training towers ensures that the rat cannot locate the “correct” tower using the uncapped food cup as a visual cue.

Figure 1. Photographs of the Open-field Tower Maze (OFTM). (A) The rat is able to find and retrieve a food reward from only one reward-baited “training tower” during acquisition, as well as all subsequent training and testing. (B) Three “pre-training towers” are placed in the center of the maze arena during pre-training. Please click here to view a larger version of this figure.

2. The OFTM Maze Room

1. Use a lamp with a 60 W light bulb (Lumens: 800) to dimly illuminate the maze room. The lamp is located 3 ft above the floor and 2 ft to the side of the maze, facing toward the ceiling. Play white noise in the background (~70 dB) using a sound machine.
2. Mount a digital video camera to the ceiling above the maze to transmit video to a computer. Hang an opaque curtain to separate the maze from the computer monitoring area.

3. Rat Handling, Food Restriction & Housing

1. Have each experimenter handle all rats for seven days (4 min per day) prior to pre-training.
2. Place rats on a food restricted diet (1 hr of free-feeding per day). This diet ensures that rats will be motivated to retrieve rewards during training, while maintaining a healthy weight (~90% of free-feeding weight).
3. Double-house rats (if using females) throughout the duration of the experiment. Pair-housed female rats are less anxious than single-housed female rats¹².

4. Pre-training Procedures

1. Bait each uncapped food cup on the seven towers with a food reward (e.g., one piece of fruit cereal) inside.
2. Place a rat into the maze in a random location until it finds and consumes all seven food rewards before it is removed.
   1. If the rat does not retrieve all of the food rewards within 10 min, then remove it from the maze and give pre-training procedures again the following day. Note: some rats will take longer than others to initially learn to retrieve the food rewards from the top of the towers.
   2. If a rat does not retrieve and consume all seven food rewards from the towers within the first two days of pre-training, give it additional 30 min sessions in the OFTM until the food rewards are retrieved. Typically, the rat will find and consume all the food rewards after one or two of these 30 min sessions.

5. Place-training & Response-training Procedures

1. Assign each rat two of four start locations. Counterbalance the following: the start location release point within and between each training day; the type of learning (place vs response); the “correct tower” for all the place learners; and the turn each response-learner is trained to make (right vs. left).
2. At the start of each training day turn on the noise machine and open the video recording program. Bait all four towers and cover three out of the four food cups, leaving the food reward on the “correct” tower exposed.
3. On each given trial, start video recording (refer to manufacturer’s user manual for instructions), then place the rat in the maze at its assigned start location. Then, go behind the curtain to observe the rat’s behavior on a computer monitor. Remove the rat from the maze immediately after it retrieves the reward or after 3 min have passed and stop video recording.
   1. For place-learning rats, always use the same “correct” food tower, and place the rat in one of two start locations. A place-learner solves the OFTM by finding a specific location using the spatial cues provided in the maze. For example, bait tower D (= the “correct” tower) and release the rat from start location ‘2’ or ‘3’ (solid arrows in Figure 2A, left), or bait tower A and release a rat from start location '1' or '4' (dashed arrows in Figure 2A, left).
   2. For response-learning rats, switch the “correct” food tower depending on the start location. Response-learners solve the OFTM by learning to make a specific turn (e.g., always turn right), regardless of its starting location in the maze. For example, bait tower D and release the rat from start location ‘3’, or bait tower B and release that same rat from a start location ‘2’ (solid arrows in Figure 2B, left). For a different rat, bait tower B and release the rat from location ‘1’, or bait tower A and release that rat from location ‘4’ (dashed arrows in Figure 2B, left).
4. Give all rats a total of 48 training trials across 12 days (four trials per day).

Figure 2. Overhead Schematic Showing Place- and Response-Training & Testing Procedures. This figure shows the start and food reward locations for training place-learners (A, Left) and response learners (B, Left), and also the start and reward locations used during the start location switch test for place-learners (A, Right) and response learners (B, Right). Numbers indicate a rat’s starting location; gray squares represent food towers; solid and dashed lines point to the baited food tower. Start locations are counterbalanced within each training condition. Please click here to view a larger version of this figure.

6. Start Location Switch Testing Procedures

1. After all the rats perform at their asymptotic level (~80% of correct first choice responding) carry out a “Start Location Switch Test” to test the persistence of the learned behavior.
2. Counterbalance the novel start locations (i.e., the start locations that have not been previously used for the rat).
3. Follow the procedures for each training trial described in protocol section 5 with the new start locations.
   1. For a place-learning rat that originally was released from location ‘2’ or ‘3’ and had tower D as its “correct” tower, place the rat in either of the new start location '1' or '4' , while still baiting tower D (Figure 2A, right).
   2. For a response-learning rat that was originally released from location ‘2’ or ‘3’ and learned to turn right to receive the reward (on towers B and D, respectively), place the rat in either of the new start location ‘1’ or ‘4’ and bait either tower A or C, respectively (which would still require the rat to turn right in order to find the food reward) Figure 2B, right.
   3. Administer all rats eight start location switch test trials across two days (four trials per day).

7. Data Analysis

1. Have an experimenter who has not trained rats and does not know the treatment of each rat score video-taped trials by counting the number of responses a rat makes to find the “correct” tower on each trial (indicated by the rat rearing up over the food cup).
2. Calculate the percent of times that a rat finds the reward on its first choice (% of first choice correct responses) within each training day. Construct learning curves using the percent of first choice correct responses across training days. Use the percent of first choice correct response for the statistical analysis.
3. Calculate the number of trials it took the rat to reach the a priori set criterion to acquire the task (i.e., first choice correct responding on 8 out of 10 consecutive trials). Exclude rats from analysis that do not reach the a priori criterion.

Results

The representative results described here have been previously published in the Neurobiology of Learning & Memory, Elsevier². In this experiment the OFTM was used to test the effects of chronic and cycling estradiol treatments on place- and response-learning in female Sprague-Dawley rats (250 – 300 g; N = 48). There were two important reasons why the OFTM was used to conduct this experiment. First, estradiol has been shown to induce differential effects on place- and response-learning⁷. Second, ...

Discussion

Numerous paradigms can be used to investigate learning and memory in rodents, and each has important steps that experimenters should follow. In the OFTM, it is important that maze conditions (e.g., position of maze, intra- and extra-maze cues, dim lighting and background noise level, daily time of training) are kept constant throughout the duration of the experiment. It is also necessary to minimize stress in rat subjects to ensure continuous performance in the OFTM. Stress can be kept to a minimum in female rat...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
OFTM construction supplies
Safety Glasses, Clear, Uncoated	Grainger	4EY97	Used for general eye protection during maze construction
Masking Tape, Beige, 18 mm x 55 m	Grainger	15F739	General supply
Wiss All-purpose Scissor	Home Depot	37103278647	General supply
Lancaster 6 foot Round Folding table - Heavy Duty white granite plastic - 29"	www.webstaurantstore.com	384YCZ72RND	Used as base of the maze
Laminate Countertop sheet in White Matte Finish (48 x 96 x 0.039)	Home Depot	1573603504896	Used as walls for the maze
Laminate Countertop sheet in Black Matte Finish (48 x 96 x 0.039)	Home Depot	1595603504896	Used as floor of the maze
Adhesive, Spray, 24 oz, 17.6 oz Net. 3M	Grainger	3MA16	Used to adhere the laminate floor to the table top and pieces of laminate to each other
16.6 oz #90 hi-strength S	Lowe's	193252	Used to adhere the laminate floor to the table top
Superflex RTV, Silicone Adhesive Sealant, Loctile	VWR	300040-068	Used to seal around the baze (between the floor and walls of the maze)
Industrial Sealant; 10.1 oz	Grainger	4UH06	Used to seal around the baze (between the floor and walls of the maze)
Painter's Masking Tape; Shurtape; 2 in x 60 yd	Grainger	6FET7	Used to mark out visual patterns on maze walls
Mini/Trim paint tray	ACE Hardware	1365105	Used to hold paint
Foam mini roller 4" 6 pk	ACE Hardware	1494871	Used for painting patterns
Val Sample Set Nbase paint	ACE Hardware	1505031	Used as paint for pattern on the walls of the maze
HP Paint Black	Walmart	72450405005	Used to paint pattern on the walls of the maze
Brush	Walmart	81773801110	Used to paint pattern on the walls of the maze
Duct Tape, 2 in x 60 yd, 10.5 mil. Black	Grainger	15F767	Used to wrap the exterior of the maze
4x4 8 ft PT pine Vinyl-CTD Post W/Cap	Home Depot	40933070541	Used to build towers for the maze
20 gram Pro Super Glue	Lowe's	125870	Used to glue tower tops, towers to the floor of the maze and food cups to the tower tops
Bead Containers 9 - 1/2	Michaels	652695766978	Used as food cups on top of towers
Spray Paint, Black, 12 oz	Grainger	6KP22	Used to paint food cups & covers
Room prepration
Conair SU1W Sound Machine; 10 Sounds	Home Depot	204493600	Used for background noise
Lamp	Walmart	5027698392	Used for dim lighting of maze room
ES 14 W DayLight Spiral Bulb 4 pk	Home Depot	762148208433	Used for dim lighting of maze room
Shower Curtain	Walmart	3429910901	Used for separation between the maze and the experimenter
Curtain Rod	Bed Bath & Beyond	76598645104	Used for separation between the maze and the experimenter
Froot Loops Cereal	Walmart	3800039122	Used as reward
Recording equipment
Media Recorder 3:4 video	Noldus	NMR4-0200	Used to record training trials
Monochrome GigE Camera	Noldus	XCFW-BG62	Used to record training trials
Precision M4800 Laptop	Dell	N/A	Used to run video software