Apr 28, 2026

Morris Water Maze: Spatial Learning and Memory Assessment in Mice

DOI
https://dx.doi.org/10.17504/protocols.io.5jyl84zk7g2w/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.5jyl84zk7g2w/v1
Protocol Citation: Sarah van Veen, Zsuzsanna Callaerts-Végh 2026. Morris Water Maze: Spatial Learning and Memory Assessment in Mice. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl84zk7g2w/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 21, 2026
Last Modified: April 28, 2026
Protocol  Integer ID: 315428
Keywords: ASAPCRN, memory assessment in mice, memory in adult mice, assessing hippocampus, morris water maze, memory consolidation, memory assessment, working memory, spatial learning, mice, adult mice, using distal spatial cue, dependent spatial learning, mice this protocol, distal spatial cue, memory
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol describes the Morris Water Maze (MWM) task for assessing hippocampus-dependent spatial
learning and memory (hidden-platform acquisition and long-term retention) and working memory in adult
mice. Mice are trained to locate a hidden platform using distal spatial cues, with probe trials conducted at
the end of each working week to assess memory consolidation.
Guidelines
•       Maintain water temperature at 26 °C throughout each day of testing.
•       Dry mice thoroughly between trials with a warm towel to prevent hypothermia.
•       Clean pool water regularly; replace ACUSOL‱ as needed to maintain opacity.
•       Exclude data for any trial in which the mouse floated or did not actively swim.
Materials
AB
SubjectsAdult WT and KO littermates (~2 months old)
Pool150 cm diameter circular pool
Water temperature26 °C (monitor with thermometer)
OpacifierACUSOL™ OP 301 Opacifier (Dow Chemicals) at 0.5% — make water opaque white
Platform15 cm diameter submerged hidden platform, 1 cm below surface
Spatial cuesDifferences in wall color and room structures (e.g., shelving, equipment) serve as distal visual cues
TrackingEthoVision video tracking equipment and software (Noldus bv, Wageningen, The Netherlands)
Inter-trial interval15–30 min between trials
Probe trial duration100 s

Before start
• Prepare the pool: fill with 26 °C water and add ACUSOL‱ OP 301 at 0.5% until water is fully opaque.
• Position the hidden platform 1 cm below the water surface in a fixed quadrant (target quadrant).
• Ensure distal spatial cues are available via differences in wall color and room structures (e.g., shelving,
equipment). Do not rearrange the room during training.
• Calibrate EthoVision tracking software: define four quadrants, record pool dimensions.
• Acclimate mice to the procedure room at least 30 min before testing.
Training trials
Each day, release each mouse from 4 different randomized starting positions (N, S, E, W quadrant
edges). For each trial, allow up to 100 s to locate the hidden platform. Once on the platform, allow
the mouse to remain for 15 s. If the mouse fails to locate the platform within 100 s, guide it to the
platform and allow 15 s there. Return mouse to a holding cage with dry bedding for 15–30 min
between trials.
Note
Starting positions should be randomized across animals and days to prevent position-based strategies.

Probe trials — end of each working week
At the end of each working week, remove the hidden platform. Release the mouse from the opposite
quadrant to the target. Allow free swimming for 100 s. Record the search pattern using EthoVision.
A preference for the target quadrant indicates spatial memory consolidation.
Working memory version
For the working memory version of the task, follow the same daily trial structure but reposition the
hidden platform to a new fixed location at the start of each day. Probe trials assess within-day
acquisition rather than long-term retention.
Data analysis
EthoVision automatically computes the following variables: path length (cm), swim velocity (cm/s),
and time spent in each zone/quadrant. Analyze escape latency and path length across training days
(learning curve). For probe trials, compare time spent in the target quadrant vs. other quadrants.
Apply appropriate statistical tests.
Expected result
WT mice should show a progressive reduction in escape latency across training days and significant preference for the target quadrant in probe trials (>25% time). Impaired spatial learning in KO mice would manifest as elevated escape latency and/or absence of quadrant preference.