Water maze FAQ: quick answers to common questions

These are the short answers. Click a question to read the full answer, the extra detail and the sources on the page where it lives.

Pool, platform and room

What size platform, and how deep below the surface?

About 10 cm across (or smaller) and 1–1.5 cm below the surface, placed halfway between the wall and the centre. Use a larger platform if you test mice in a rat pool. Your local ethics committee, licence conditions and vet always take precedence.

How deep should the pool and the water be?

The water must be deep enough that the animal cannot touch the bottom, with enough wall above it to stop escapes. Rats: about 25–30 cm of water with about 25–30 cm of wall above. Mice: shallower, so they can see over the wall to the room cues. Your local ethics committee, licence conditions and vet always take precedence.

Which pool colour or dye should I use for my animals' coat colour?

Pick the opposite of the coat: a dark pool (or dark, clear water) for white or albino animals, and white water or a white pool for black or dark animals. Use beige if you test both. Your local ethics committee, licence conditions and vet always take precedence.

Where should I put the platform in the water maze?

The platform is the small escape platform the animal swims to in the water maze; it is hidden just under the water so the animal has to remember where it is. Put it in the middle of one quadrant, halfway between the pool wall and the centre, not in the centre and not against the wall, with its top about 1–1.5 cm below the surface. 1) Keep it in the same place for every learning (acquisition) trial. 2) Take it out for a probe trial. 3) Move it to the opposite quadrant for reversal. 4) Tell HVS Image where it is: on the calibration screen, calibrate the pool centre first (platform 0), then each platform position (1–4, platform 1 is the north-east quadrant), or click 'Suggested MWM Platform Positions' to fill in one position per quadrant.

How often should I change the pool water?

Remove faeces daily and change the water once or twice a week (more often if you add paint or milk). Refill on the same day so it settles to room temperature overnight. Your local ethics committee, licence conditions and vet always take precedence.

How should I measure water temperature?

Stir the water, then measure at the same depth and place each time (for example just below the surface near a start point) before each session. Record it. Your local ethics committee, licence conditions and vet always take precedence.

How bright should the testing room be?

There is no single correct level. Choose one for your test, measure it with a lux meter at the animal's level, keep it the same every day and report it. Open field protocols often use bright light (500–1000 lux); water maze rooms are usually moderate. Your local ethics committee, licence conditions and vet always take precedence.

How do I stop reflections and glare on the water spoiling tracking?

You stop reflections by lighting the pool indirectly. 1) Use uplighters, or bounce light off the ceiling or walls, rather than lamps shining down at the water. 2) Look at the camera image: there should be no white patches on the water. 3) Check the light is even across the pool. Your local ethics committee, licence conditions and vet always take precedence.

Cues

Where should I put the distal cues, and why?

Put several large, distinct cues on the walls around the maze, high enough to be seen over the pool wall, and never move them during a study. Animals use them to work out where the platform or escape hole is. Make the cue set asymmetric so that no two directions look the same. Your local ethics committee, licence conditions and vet always take precedence.

How big and what colour should cues be?

Use big, bold, high-contrast shapes (black and white work best). Rodents see detail poorly and do not see red, so colour differences that you notice may not help them. Your local ethics committee, licence conditions and vet always take precedence.

Why should I run a cued (visible platform) trial?

A cued trial checks that each animal can see and swim well enough and is motivated to escape. If it fails the cued trial, a poor hidden-platform result may not mean poor memory. Your local ethics committee, licence conditions and vet always take precedence.

What uncontrolled cues could my animals be using?

Your animals could be using anything stable that they can see, hear or smell, such as doors, shelves, lights, the camera, curtains, heaters, cages, the experimenter and even a noisy vent. These are fine as long as they never move. Walk round the room at the animal's eye level and list them. Your local ethics committee, licence conditions and vet always take precedence.

Can the tracking camera or lights become a cue?

They can if they change. Keep the camera, PC and lights in the same place and state for every trial, and cover any indicator LEDs the animal could see. Your local ethics committee, licence conditions and vet always take precedence.

Animals

Which mouse strain is best for the water maze or Barnes maze?

Choose a strain with normal vision, and check what is known about it in your test. C57BL/6J and 129 substrains are commonly used; avoid strains that are blind as adults (for example C3H/HeJ, CBA/J, FVB/NJ) for visual spatial tasks. Your local ethics committee, licence conditions and vet always take precedence.

Which rat strain should I use?

Most common rat strains can learn the water maze, so choose one that suits your research question and keep it consistent. Many strains work, including Sprague Dawley, Long Evans, Lister, F344, Wistar and Dark Agouti. Pigmented rats see better than albino rats, but albino rats can still do the water maze well. Your local ethics committee, licence conditions and vet always take precedence.

How should I handle and habituate rats?

You should handle rats gently for several days before testing, so they are calm with you. Tickling (about 15 seconds a day for 3 days) can help them become relaxed with you. Stop if the rat avoids your hand. Your local ethics committee, licence conditions and vet always take precedence.

How does stress affect maze results?

Stress can make animals learn worse, hug the wall or swim slower, which looks like a memory problem when it is not. Keep handling, temperature, light, noise and timing the same for every animal. Your local ethics committee, licence conditions and vet always take precedence.

Can the experimenter affect the results?

Yes. Who handles the animals, and how, can change behaviour. Use the same person (or balance people across groups), record who ran each animal, and keep them blind to group where you can. Your local ethics committee, licence conditions and vet always take precedence.

How long should animals settle in the testing room before a trial?

Bring animals into the testing room in their home cages and let them settle before you start: at least 30 minutes in published protocols. Your vet or a competent person sets the acclimatisation period after arrival at the facility. Your local ethics committee, licence conditions and vet always take precedence.

Measures

What are the limitations of escape latency?

Escape latency is the time the subject takes to reach the platform. Latency doesn't show strategy and is affected by speed, start distance and floor or ceiling effects, so read it with path ratio, heading, Gallagher measures and behaviour identification. HVS starts timing when you click as the subject enters, and ends it by platform detection, after a dwell time, or manually.

When is percentage time in the target quadrant a good measure?

In probe trials, a preference for the target quadrant regardless of start point indicates spatial memory, but only when platforms sit at quadrant centres. Otherwise use close passes and Gallagher measures. Time slices reveal early preference.

Why can platform crossings be misleading in probe trials?

Whole-trial crossings favour subjects that keep returning (perseverating) over flexible ones that check and move on. Use heading, the first close encounter, time slices and behaviour identification as well.

How do I interpret thigmotaxis, and how do I compare thigmotactic subjects fairly?

Thigmotaxis is the tendency of an animal to stay close to the walls of an arena, often called 'wall-hugging'; in the water maze it means swimming in the outer ring next to the pool wall instead of searching the open water. A little wall-hugging in the first trials is normal while animals adapt; if it carries on, it may reflect stress or anxiety, not understanding the task, a sensory or motor problem, or low motivation. HVS Image reports the percentage of time and of path spent in a band next to the wall. To compare wall-hugging animals fairly, use heading angle, time slices and Gallagher by segment rather than whole-trial Gallagher or close-area measures.

How should I interpret high inactivity?

High inactivity means the subject spent a lot of the trial floating or moving very slowly. It can come from tiredness, stress, low motivation, a drug effect, a movement problem or poor memory, so it is not a learning score on its own. 1) Compare it with latency and path length. 2) Check crossings, close encounters and Gallagher proximity. 3) Good proximity with high inactivity suggests the subject still knows where the target is.

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For a single page on every common measure, see HVS Image's guide to Morris water maze analysis.