Protocol for Water Maze
Materials Required
Principle
The Morris Water Maze is a rodent spatial learning and memory assay in which a mouse or rat swims in opaque water to find an escape platform; in the hidden-platform version, the animal cannot see the platform and must use distal extra-maze cues to learn its fixed spatial location[1][2][3][4].
The assay primarily measures hippocampus-dependent spatial learning during acquisition trials and spatial reference memory during probe trials after platform removal; readouts include escape latency, swim path length, swim speed, quadrant occupancy, platform-site crossings, and proximity to the former platform location[2][3][4][5][6].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Clean water is used as the swimming medium, and the water is made opaque with a non-toxic opacifying agent such as powdered non-fat milk or non-toxic tempera paint so that the animal cannot see the hidden platform[3][7].
• Mice or rats are used depending on the study design; species, strain, sex, age, genotype, vision, motor ability, and disease model should be reported because these factors can affect swimming, learning, and interpretation[3][4][8][9].
• Mice or rats are used depending on the study design; species, strain, sex, age, genotype, vision, motor ability, and disease model should be reported because these factors can affect swimming, learning, and interpretation[3][4][8][9].
Equipment and instruments
• Use a circular water tank, escape platform, extra-maze visual cues, video camera, computerized tracking system or blinded scoring method, timer, water-temperature monitoring, and drying or warming materials for post-trial recovery[3][4][7].Controls
• Use wild-type, sham, vehicle-treated, or non-impaired controls appropriate to the model; include visible-platform or cued-platform trials when needed to control for visual, motivational, or motor confounds[3][4][7][8].Experimental Procedure
Preparation Steps
• Set up the circular pool with opaque water, place stable distal visual cues around the testing room, and keep room layout, lighting, platform position, water opacity, and tracking settings constant across acquisition days[2][3][4][7].• Prepare the hidden platform so that it is submerged below the opaque water surface during spatial acquisition trials; prepare a visible platform condition when assessing vision, swimming ability, motivation, or non-spatial learning[3][4][7].
• Assign animals randomly to test order when feasible, keep handling and inter-trial conditions consistent, and define exclusion criteria before testing for animals unable to swim or perform the visible-platform control[3][4][8].
Operation Steps
• For hidden-platform acquisition, release the animal into the pool from predefined start positions and allow it to search for the submerged platform; if the animal finds the platform, allow it to remain briefly before removal according to the selected protocol[3][4][7].• Repeat acquisition trials across multiple trials and days; published protocols vary, including multi-day protocols that can generate results in approximately 6 days and shorter 2-day mouse protocols designed for specific applications, so the exact trial number, inter-trial interval, and training duration should be selected from the cited protocol and reported explicitly[3][8].
• For probe testing, remove the platform after acquisition and allow the animal to swim while recording spatial search bias for the previous platform location[3][5][6][7].
• For visible-platform testing, place the platform in a visible condition and analyze escape performance to identify visual, sensorimotor, or motivational impairments that could confound hidden-platform learning[3][4][7][8].
• After each trial, remove and dry the animal and maintain consistent recovery conditions before the next trial[3][7]• Data Acquisition and Analysis
For acquisition learning, collect escape latency, path length, swim speed, and search strategy or trajectory measures; path length and latency should be interpreted together with swim speed because motor differences can affect latency independently of spatial learning[3][4][6][8].
• For probe trials, quantify target-quadrant time, platform-location crossings, time in annulus, and proximity to the former platform location; proximity-based measures have been reported to be more sensitive than several traditional probe measures in detecting group differences[5][6].
• Use the individual animal as the biological replicate, analyze repeated acquisition trials with appropriate repeated-measures or mixed-model statistics when applicable, and report species, strain, sex, age, group size, pool size, water temperature, platform size and position, cue arrangement, trial duration, probe duration, tracking method, blinding, and exclusion criteria[3][4][8][9].
• Interpret impaired hidden-platform performance only after checking visible-platform performance, swim speed, and gross motor or visual ability[3][4][7][8].
Troubleshooting
Problem: Animals show poor hidden-platform learning.
Possible Cause: Spatial cue configuration, platform position, strain background, or protocol design may not support reliable learning.Literature-supported Solution: Use stable distal visual cues, keep the platform location constant during acquisition, and optimize the protocol for the strain and testing environment[2][3][4][9].
Problem: Escape latency differs between groups, but swim speed also differs.
Possible Cause: Motor impairment or altered swimming performance may confound latency-based learning measures.Literature-supported Solution: Analyze path length, swim speed, and visible-platform performance in addition to latency[3][4][7][8].
Problem: A group performs poorly in both hidden- and visible-platform trials.
Possible Cause: Visual, motivational, or sensorimotor deficits may prevent valid interpretation of spatial learning.Literature-supported Solution: Use visible-platform or cued-platform testing to identify non-spatial performance confounds before concluding hippocampal-dependent spatial memory impairment[3][4][7][8].
Problem: Probe-trial results are weak despite acquisition differences.
Possible Cause: Some traditional probe measures may be less sensitive to spatial memory differences.Literature-supported Solution: Include proximity to the former platform location along with quadrant time and platform crossings[5][6].
References:
- [1]. Morris RGM. Spatial localization does not require the presence of local cues. Learn Motiv. 1981;12(2):239-260.
- [2]. Morris RGM, et al. Place navigation impaired in rats with hippocampal lesions. Nature. 1982;297(5868):681-683. [Content Brief]
- [3]. Vorhees CV, et al. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc. 2006;1(2):848-858. [Content Brief]
- [4]. D’Hooge R, et al. Applications of the Morris water maze in the study of learning and memory. Brain Res Brain Res Rev. 2001;36(1):60-90. [Content Brief]
- [5]. Maei HR, et al. What is the most sensitive measure of water maze probe test performance? Front Integr Neurosci. 2009;3:4. [Content Brief]
- [6]. Vouros A, et al. A generalised framework for detailed classification of swimming paths inside the Morris Water Maze. Sci Rep. 2018;8(1):15089.
- [7]. Bromley-Brits K, et al. Morris water maze test for learning and memory deficits in Alzheimer’s disease model mice. J Vis Exp. 2011;(53):2920. [Content Brief]
- [8]. Gulinello M, et al. Validation of a 2-day water maze protocol in mice. Behav Brain Res. 2009;196(2):220-227. [Content Brief]
- [9]. Weitzner DS, et al. Morris water maze test: optimization for mouse strain and testing environment. J Vis Exp. 2015;(100):e52706. [Content Brief]
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