Documentation

Introduction

The Pool Shape Manipulation assay is a modification of the Morris Water Maze task that is applied in the evaluation of spatial navigation and memory capabilities of rodents. The Pool Shape Manipulation allows assessments wherein the focus is on the spatial information of the maze from other features of the environment except for extra maze cues. The paradigm is applied using two Morris Water Mazes, one with a square insert and another a simple circular pool.

The studies have shown that mazes of different geometry affect the spatial navigation of rodents. Lee, Tucci, Sovrano, and Vallortigara’s (2015) study revealed the successful utilization of boundary geometry in a working memory task by rodents when evaluated in a square and a rectangular arena. Another study conducted by Yaski, Portugali, and Eilam (2011) utilized a square and a circular arena and found that the rodents showed a significant difference in the path shape between the arenas. Most studies of the effects of global geometry often utilize dry mazes and appetitive motivations. However, the Pool Shape Manipulation assay relies on the innate aversiveness of rodents to water. The added stress factor allows observation of the shifts in navigation behaviors and decision making of rodents. The assay can also be extended to evaluate the effects of lesions, drug testing, and diseases and disorders that affect memory. 

Other available Morris Water Maze manipulations and modifications include the Y-maze Insert, Star Maze Insert, Radial Arm Maze Insert, Snow Cone Insert, and Plus Maze Insert.

Apparatus and Equipment

The Pool Shape Manipulation assay uses an opaque circular pool measuring 127 cm in diameter. An opaque square inlay measuring 90 × 90 cm allows conversion of the pool into a square pool. The pool and the inlay both have a height of 48 cm. A square escape platform measuring 31 cm in height is also included.

Training Protocol

Clean the apparatus before and after trials or as needed to prevent the influence of any lingering stimuli. Ensure that the apparatus is appropriately lit, and the task is performed in an undisturbed environment to minimize the influence of any external stimuli on the performance. Tracking and recording of the performances can be done with the assistance of an external tracking and video system such as the Noldus EthoVision XT.

Protocols will vary depending on the investigatory needs of the experiment. The following is a sample protocol for the evaluation of spatial navigation behaviors using the Pool Manipulation Assay.

Maintain the water temperature and extra maze cues throughout the experiments. In order to ease observations, divide the pool, virtually, into four equal-sized quadrants indicated by cardinal directions (North, South, East, and West). Fill the maze with water such that the platform is hidden 2 cm under the water level.

Pretraining (Phase I)

Train the subjects in the circular pool or the square pool. Place the hidden platform in one of the quadrants and maintain the position throughout the training. Choose at random a starting point from one of the eight starting points in the quadrants. Place the subject facing the wall and allow it to explore the arena for 60 seconds. If the subject fails to find the platform, remove it from the water and place it on the platform. Allow the subject to stay for 10 seconds on the platform before ending the trial. Hold the subject in its home cage between inter-trial intervals. Perform 8 trials per session per day for four days.

Phase II

Probe Trials

Use the square pool if the subject was trained in the circular pool and vice versa. Remove the hidden platform and introduce the subject into the maze facing the wall. Allow it 30 seconds to find the platform.

Training Trials

Maintain the pool shape used in the probe trials. Place the hidden platform in the quadrant opposite the one used in the pretraining trials and maintain its position throughout the training. Begin trial by placing the subject in one of the 15 potential starting points and perform trials for 2 hours. Use an appropriate inter-trial interval.

Retention Trial (Phase III)

Perform retention trial 24 hours after the completion of Phase II. Maintain the pool shape used in the training trials of Phase II. Remove the platform from the arena and observe the performances of the subject in a 30 seconds trial.

Literature Review

Investigation of the effects of pool shape manipulation of spatial memory

Bye, Hong, Moore, Deibel, and McDonald (2019) investigated the performances of male Long Evans rats in a Pool Shape Manipulation assay. The subjects were divided into two groups: The same group and New group. All training and testing were performed in a 4.5 × 3 m room, with geometric-shaped posters on the white walls, a computer, an upright storage shelf along with the experimenter serving as the extra maze cues. The positions of the pools were consistent throughout the investigation. Half of both groups were trained in the square pool, while the other half was trained in the circular pool. During the 4 days training period, a significant reduction in the latencies to find the platform and path lengths were observed for both groups, with no significant difference in group-based performances. Following pretraining, the New group underwent phase II using the other pool shape while the Same group was evaluated using the same pool used in the pretraining phase. Groups were mass-trained for the second part of phase II. The probe trials revealed spatial preference in the Same group, which was absent in the New group. The Same group was observed spending more time in the target quadrant than the other quadrants. Both groups were observed to display significant improvement in performances as the trials progressed in the second half of phase II. At the end of the training session, path lengths had reduced to 1.7 m and 1.4 m for the Same and New group, respectively, which was a significant reduction from 8.7 m and 9.0 m first-trial path lengths. Retention trial performed 24 hours after phase II revealed no significant performance differences suggesting that both groups were able to form a rapid memory of the new platform location.

Data Analysis

The following behaviors and parameters can be recorded using Pool Shape Manipulation assay

• Latency to initiate the task
• Latency to reach the hidden platform
• Time spent in a quadrant
• Frequency of visit to a quadrant
• Number of correct quadrant entries
• Percentage error
• Heading error
• Path length

Strengths and Limitations

Strengths

The Pool Shape Manipulation assay allows observations on how maze shape affects navigation and navigation memory in rodents. Different visual cues can be attached to the walls of the square insert, which, along with the movable escape platform, can be used to evaluate working and reference memory. Since the maze task relies on aversive stimuli, innate fear of drowning, additional stimulation is not required.

Limitations

Rodents find water aversive, and hence the Pool Shape Manipulation task can be quite stressful for the animals. Additionally, the task is not suitable for animals with motor impairments since it relies heavily on swimming ability. Changes in the water temperatures during testing can also influence task performances. Unnecessary or improper handling of the subjects, in addition to unintended stimuli, can affect performances and observed behaviors. Individual differences, as well as the gender, strain, species, and age of the subjects, may contribute to the observed behaviors.

Summary

• The Pool Shape Manipulation assay is a modification of the Morris Water Maze.
• The apparatus is used in the assessment of the effects of pool shape on navigation behaviors.
• The assay includes a square pool and a circular pool to evaluate learning and memory in rodents.
• The apparatus comes with a removable and movable escape platform.
• Water is an aversive stimulus, and hence, the Pool Shape Manipulation assay can be stressful for rodents.
• The assay is not suitable for animals with locomotor deficits.

References

1. Bye, C. M., Hong, N. S., Moore, K., Deibel, S. H., & McDonald, R. J. (2019). The effects of pool shape manipulations on rat spatial memory acquired in the Morris water maze. Learning & behavior, 47(1), 29-37.
2. Lee, S. A., Tucci, V., Sovrano, V. A., & Vallortigara, G. (2015). Working memory and reference memory tests of spatial navigation in mice (Mus musculus). Journal of Comparative Psychology, 129(2), 189.
3. Yaski, O., Portugali, J., & Eilam, D. (2011). Arena geometry and path shape: When rats travel in straight or in circuitous paths? Behavioural brain research, 225(2), 449-454.