Morris water maze protocol for mice and rats

The Morris water maze tests spatial learning. A rat or mouse swims from points around the edge of a round pool to a platform hidden under the water, using distant cues around the room.¹ Learning shows up over repeated trials, and memory shows up as a preference for where the platform was once it is taken away.¹ Richard Morris described further developments of the procedure in 1984, with rats learning to escape from opaque water onto a hidden platform.²

D’Hooge and De Deyn call it one of the most used tools in behavioral neuroscience.³ Escape from water depends little on activity or body weight, which makes the test useful for many kinds of animal model.¹ Vorhees and Williams note that performance is strongly correlated with synaptic plasticity in the hippocampus.¹ Damage to the hippocampus, striatum, basal forebrain, cerebellum or cortex can impair it.³

Scores also depend on the pool, the training plan and the animals themselves, including sex, strain, age, diet, stress and infection.³ Vorhees and Williams trace most of the test’s weak points to pools that are too small, protocols that do not test learning well enough, probe trials given too soon and missing control trials.¹

Pool size sets how hard the task is. The search area grows with the square of the radius, so a bigger pool sets a much harder problem.¹ A pool that is too small lets animals find the platform by other means, such as swimming a set distance from the wall, with little use of the room cues.¹

In Vorhees and Williams’ comparison, rats in a 122 cm pool leveled off by the second day. Rats in a 210 cm pool kept improving up to day 4 and leveled off on day 5.¹ They do not recommend a 122 cm pool for rats, though it is the most common size for mice and gives good learning curves.¹

Check a pool for seams, ridges or marks on the inside, which give the animal proximal cues close at hand. Swimming pools and troughs sold for other uses often have them.¹ Smooth walls also cut down on climbing in early trials.¹

Start with a platform about 10 cm across. Vorhees and Williams found that starting with a smaller one makes the task too difficult, so keep small platforms for later phases.¹ Give the platform a wide base so it does not tip or slide when an animal climbs on or bumps it, and a textured top for grip.¹

Hide the platform by making the water opaque with non-toxic tempera paint or powdered milk.⁵ For white mice, Bromley-Brits and colleagues use a black pool with clear water and a clear platform instead.⁴

Animals find the platform from cues around the room, so the room needs plenty of them. When in doubt add more, since there is no evidence that too many cues cause problems, while too few may.¹ Put high-contrast cues around the room.⁴ Never move the cues during testing, because they are the animal’s reference points.¹ Hang curtains around the pool. Closing them hides the room cues during cued trials, a control described below.¹

If you stay in view, you become one of the cues. Leave the room, stand behind a screen or stay in one fixed spot, and never move around while the animal swims.^1,4

Use indirect light. Tracking software picks up reflections from the water, and indirect light keeps them down.¹ If you film the trials, keep the whole pool in the camera’s view.⁵

Rats perform well in water at room temperature, 19 to 22 °C, with no sign of fatigue or hypothermia at the usual test ages.¹ Mice may be more prone to hypothermia that affects their performance. Even so, water at 20 to 22 °C generally reduces floating in mice.¹ Bromley-Brits and colleagues use 22 °C for mice, and Nunez uses 26 °C for rats. Whatever you choose, keep it within a degree across animals and days.^4,5

Water left to reach room temperature can take one to three days, or you can add hot water to speed it up.⁴ Bring the animals into the room at least 30 minutes before testing, and keep them where they cannot see the pool or the cues.⁴ Dry each animal after its trials and make sure it is warm before it goes back to the animal facility.⁴

These steps follow Vorhees and Williams’ hidden platform protocol. The platform stays in the middle of one quadrant, halfway between the center and the wall.¹

1. Plan the start points

   Name four start points around the edge, N, S, E and W, with S at your own position.¹ Use each one once a day, in an order that changes so the animal cannot learn a fixed set of turns.¹

2. Place the animal facing the wall

   Lower the animal into the water at the start point, tail first and facing the pool wall. Never drop it in.^1,5 Start the timer the moment you let go.¹

3. Stop the timer at the platform

   Stop timing when the animal reaches the platform. The usual limit is 1 minute for mice and 2 minutes for rats.¹

4. Guide animals that miss it

   If time runs out, place the animal on the platform or guide it there.¹

5. Leave it on the platform

   Leave the animal on the platform for about 15 seconds. In the usual design those 15 seconds are the gap between trials, and that gap gives good learning.¹ Mice often learn better with longer gaps, especially in the first session.¹

6. Repeat from a new start point

   Give four trials a day, one from each start point.¹ For mice, run trial 1 for every animal before starting trial 2, which improves learning.¹

7. Repeat on the following days

   Five to six days of four trials is usually enough for rats in a 210 cm pool, or mice in a 122 cm pool, to level off.¹

With four trials a day, the first day takes 5 to 10 minutes per animal, and less each day after that.¹ Some designs fit training into one day. Nunez gives rats three training trials to a visible platform, then 12 hidden platform trials in one day, because hormone levels in females cycle over days.⁵

Balance age, sex and strain across groups. Aged mice tend to perform poorly in the water maze, and male rodents tend to do better than females.⁴ Keep temperature, light and noise the same throughout, because stressed animals perform worse.⁴

Take the platform out, then start each animal from a point it never started from in training, such as the edge directly opposite where the platform was.^1,4 Run the probe for 30 seconds, because preference for the target quadrant fades after that.¹

Give the probe 24 hours after the last training day. A probe right after training measures short-term recall rather than long-term memory.¹ Keep extra probes during training to a few, because each one is an extinction trial and can slow learning.¹

The most used measure is the percent of time or distance in the target quadrant, where chance is 25%.¹ Average distance to the platform site and the first bearing also hold up well. Crossings of the old platform site vary more and happen less often, especially in a large pool.¹

To look harder for spatial deficits, run a reversal. Move the platform to the opposite quadrant, train for 5 more days, then give a second probe 24 hours later.¹ In reversal, mice tend to swim to the old spot first and then search in an arc, while rats switch quickly to the new spot.¹

Vorhees and Williams describe a factor analysis of about 1,500 mice across strains, in which memory accounted for only 13% of the variation in performance.¹ Wall hugging (thigmotaxis) accounted for 48%, and passivity, meaning slow swimming or floating, for 20%.¹ With much of the variation unrelated to learning and memory, controls matter especially in mouse studies.¹

Cued trials check that an animal can see, swim and climb onto the platform. Close the curtains, mark the platform with a flag about 12 cm above the water, and move the platform and start point on every trial.¹ If an animal cannot do the cued task, its spatial results are in doubt. Vorhees and Williams note that this control is too often left out.¹ Running cued trials first has advantages, especially for mice, and it cuts down on jumping off and swimming over the platform.¹

Bromley-Brits and colleagues open their mouse protocol with a day of visible platform trials, a form of cued trial. A group difference that day likely points to vision rather than memory, and they analyze the later days only when there is none.⁴

Check swim speed as well, from the training trials or in a straight swim channel before the maze. Vorhees and Williams use a channel 15 by 244 cm with an escape platform at one end.¹

Average each day’s four trials into a block, and also look trial by trial to see learning within each day.¹ A shallower curve points to a deficit in learning the task.⁴

Compare the groups on trials 1 and 2 of day 1. A difference there warns that the groups started apart, which can cloud the learning curves.¹ Analyze the days with group and day as factors. Learning data rarely meet the sphericity assumption of a repeated measures ANOVA. Test for it and adjust the F-tests, or fit a mixed model.¹

• Mice float instead of swimming. Floating is more common in mice than rats, and 20 to 22 °C water reduces it.^4,1 Prodding or startling a mouse is hard to apply evenly, so use a fixed rule instead. Vorhees and Williams give a floater up to four tries over two days, then remove it and compare floater rates between groups.¹
• Animals hug the wall. A lot of wall swimming, especially in rats, suggests the animal has not learned that the edge has no exit. Run cued trials first.¹
• Animals reach the platform, then jump off and keep searching. Running cued trials first teaches them that the platform is the way out.¹
• Rats learn in a day or two. The pool is likely too small. Vorhees and Williams use 210 cm for rats and do not recommend 122 cm.¹
• Groups differ on the first trials or the cued day. Check vision, swimming and early performance before you read the learning curves as memory.^1,4
• Tracking loses the animal. Switch to indirect light to cut reflections from the water.¹
• A mouse strain floats or turns in circles. Some strains cannot be used, and many labs report that C57BL mice are among the best performers.¹