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Morris water maze assessment of spatial learning and memory in mice

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Assess spatial learning and memory in adult mice using hidden-platform training and platform-free probe trials, with escape latency, path length, swim velocity and target-quadrant time as readouts.

Scope and method

Sample：Live adult mice

Learning and Memory Assessment

Bill of materials (BOM)

StepMaterialRoleAmountRequired / optionalSubstitution conditionsSKU / specificationLiterature product
Training trialsTowels for drying experimental animalsDry animals after removal from the water between maze trials, following the warming arrangements in the applicable protocol.

The source guidelines require a warm towel for drying animals; material, dimensions and quantity per animal are not reported.

Not reported in the sourceRequiredNot reported in sourceAvailable for inquiryNot reported in source
Training trialsDry bedding for a water-maze holding cageReturn animals to a holding cage with dry bedding between trials.

The source explicitly requires dry bedding but does not report bedding material, mass or quantity per animal.

Not reported in the sourceRequiredNot reported in sourceAvailable for inquiryNot reported in source
Training trialsACUSOL OP301 opacifierWater body shading

ACUSOL OP301; the water body is uniform and opaque white.

0.5%RequiredNot reported in sourceAvailable for inquiryNot reported in source
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Protocol #188 · revision 12 Towels for drying experimental animals | Not reported in the source | Available for inquiry Dry bedding for a water-maze holding cage | Not reported in the source | Available for inquiry ACUSOL OP301 opacifier | 0.5% | Available for inquiry

Procedure

  1. Training trials

    Release each mouse from 4 different starting positions at the edges of the N, S, E, and W quadrants each day. Allow up to 100 s to find the hidden platform in each trial and let the mouse remain on the platform for 15 s. If it fails to find the platform within 100 s, guide it to the platform and allow it to remain for 15 s. Between trials, return the mouse to a temporary holding cage with dry bedding for 15–30 min. Randomize starting positions between animals and across training days to avoid a fixed-location strategy.

  2. Platform-free probe trial

    At the end of each working week, remove the hidden platform and release the mouse from the quadrant opposite the former platform location, allowing free swimming for 100 s. Record the search path using EthoVision. Preference for the target quadrant reflects consolidation of spatial memory.

  3. Data analysis

    Use EthoVision to calculate path length (cm), swimming speed (cm/s), and time spent in each region or quadrant automatically. Analyze escape latency and path length across training days to generate learning curves. In the probe trial, compare time in the target quadrant with time in other quadrants, using statistical tests appropriate to the study design. Escape latency in WT mice should decrease with training, and target-quadrant preference in the probe trial should reach >25% of time. KO mice with spatial learning impairment may show prolonged escape latency or lack of quadrant preference.

Quality control and readouts

The pool is 150 cm in diameter and the water temperature is 26 °C; the platform is 15 cm in diameter and 1 cm below the water surface. Make the water opaque white using 0.5% ACUSOL OP301. Record swimming speed simultaneously and interpret performance together with path length and time spent in the target quadrant.

Escape latency, path length, and swimming speed during training; time in the target quadrant and search paths during the probe trial.

Study design
After repeated training, remove the platform for a probe trial and compare learning curves during training and target-quadrant preference during the probe trial.
Randomisation
Each day, release animals from the four N, S, E, and W starting positions, with the starting positions randomized across animals and training days.
Blinding
Blind experimental procedures and data analysis to group allocation.

References

  1. Sarah van Veen; Zsuzsanna Callaerts-Végh. Morris Water Maze: Spatial Learning and Memory Assessment in Mice. protocols.io, version 1, 2026-04-28. DOI:10.17504/protocols.io.5jyl84zk7g2w/v1. · DOI: 10.17504/protocols.io.5jyl84zk7g2w/v1
  2. Pascual-Lucas Maria, Viana da Silva Silvia, Di Scala Marianna et al. Insulin-like growth factor 2 reverses memory and synaptic deficits in APP transgenic mice. 2014. DOI:10.15252/emmm.201404228; PMID:25100745. · DOI: 10.15252/emmm.201404228
  3. Salas Isabel H., Callaerts-Vegh Zsuzsanna, D’Hooge Rudi et al. Increased Insoluble Amyloid-β Induces Negligible Cognitive Deficits in Old App NL / NL Knock-In Mice. 2018. DOI:10.3233/JAD-180410; PMID:30320577. · DOI: 10.3233/JAD-180410
  4. Wang Lu, Guo Lan, Lu Lin et al. Synaptosomal Mitochondrial Dysfunction in 5xFAD Mouse Model of Alzheimer's Disease. 2016. DOI:10.1371/journal.pone.0150441; PMID:26942905. · DOI: 10.1371/journal.pone.0150441
  5. Corbett Brian F., You Jason C., Zhang Xiaohong et al. ΔFosB regulates gene expression and cognitive dysfunction in a mouse model of Alzheimer’s disease. 2017. DOI:10.1016/j.celrep.2017.06.040; PMID:28700937. · DOI: 10.1016/j.celrep.2017.06.040
  6. Hinrich Anthony J, Jodelka Francine M, Chang Jennifer L et al. Therapeutic correction of ApoER2 splicing in Alzheimer's disease mice using antisense oligonucleotides. 2016. DOI:10.15252/emmm.201505846; PMID:26902204. · DOI: 10.15252/emmm.201505846
  7. Wang Zhi-Hao, Gong Ke, Liu Xia et al. C/EBPβ regulates delta-secretase expression and mediates pathogenesis in mouse models of Alzheimer’s disease. 2018. DOI:10.1038/s41467-018-04120-z; PMID:29725016. · DOI: 10.1038/s41467-018-04120-z
  8. Bailey et al. Effects of the C57BL/6 strain background on tauopathy progression in the rTg4510 mouse model. Molecular Neurodegeneration 2014;9:8. DOI:10.1186/1750-1326-9-8; PMID:24428919. Model/MWM applicability; the adopted procedure remains the separate protocols.io source version. · DOI: 10.1186/1750-1326-9-8
  9. Bouter et al. N-truncated amyloid beta (Abeta) 4-42 forms stable aggregates and induces acute and long-lasting behavioral deficits. Acta Neuropathologica 2013;126:189–205. DOI:10.1007/s00401-013-1129-2; PMID:23685882. Model/MWM applicability; source-specific assay parameters are not claimed to reproduce this article. · DOI: 10.1007/s00401-013-1129-2
  10. PMID:40906458. Methods and Materials; Figure7 and APOE4 Aggregated Cognitive Deficits Induced by Blood-Derived Aβ. Humanized APOE4 recipients after marrow transplantation; the phenotype is not attributed to APOE4 genotype alone. · PMID: 40906458

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