Why each part of the water maze matters

Good lighting needs adequate light, no specular reflection and even illumination. Shadows and glare can pull the tracking cross away from the subject, producing jumps and straight lines that change path length, speed and every location measure. Keep lighting unchanged throughout an experiment so trials stay comparable.

Lighting is also an experimental variable, not only a tracking one: in BALB/c mice, bright light during testing increased thigmotaxis and impaired water maze performance, and corticosterone and thigmotaxis correlated with latency (Huang et al., 2012). Report your lighting and keep it constant across groups.

Position the camera so the tracking area almost fills the view. Changing camera settings mid-experiment breaks consistency with the calibration, so set up and test with dummy trials before live trials.

Because all spatial measures are scaled from the calibration, any change of camera height, lens or zoom after calibrating alters the pixel-to-centimetre relationship. If a change is unavoidable, recalibrate and note it in your records.

Distal extramaze cues support allocentric, hippocampal-dependent navigation. Cues on or inside the pool wall act as local landmarks, which can compensate for impairments and change learning rates and comparability, so keep the cue arrangement consistent within a study.

Morris (1981) showed that rats can locate a hidden platform from distal cues without local cues, and Vorhees and Williams (2014) describe the allocentric (hippocampal) versus egocentric (dorsal striatal) distinction. A highly salient distal cue array was needed for spatial strategy selection in mice (Rogers et al., 2017).

A non-rising platform allows non-spatial strategies, so falling latencies can be misread as spatial learning, and in probes strong learners may leave once the platform has gone. The Atlantis sits well below the surface and rises when the subject reaches or dwells at the target.

Use the rising platform to make sure spatial learning is needed, a fixed one to see which strategy animals prefer, and both to learn preference and capability. Whichever you use, go beyond latency and target-quadrant time: heading, Gallagher proximity, close passes, pool circling and time slices.

Reference memory is shown by a preference for the platform area when the platform is absent (Vorhees and Williams, 2006). Set auto-end to No and choose a fixed ‘Max Seconds’, so every probe lasts the same time.

Maei et al. (2009) found that proximity to the former platform outperformed quadrant, zone and crossing measures across more than 1600 mouse probe tests. Blokland et al. (2004) found that quadrant measures may overestimate spatial ability and that annulus time changes within a 2-minute probe, so time slices are worth using.