The testing room

Animals notice more about the testing room than we do: the hum of a fridge, a smell from the last animal, a change in where the experimenter stands. These questions cover the room itself and the small routines that keep it the same from day to day.

Questions people ask

How should I set up the testing room?

Use a quiet room kept only for testing, with the maze in the middle and plenty of fixed cues on the walls. 1) Keep the tracking PC and the experimenter out of the animal's view, behind a curtain or screen. 2) Keep everything in the same place for the whole study. 3) Keep the room at a steady temperature (usually 20–24 °C). Your local ethics committee, licence conditions and vet always take precedence.

More detail

Animals navigate by cues round the room, so anything that moves between trials (people, bags, trolleys) can become a misleading cue. Hide the experimenter with drapes or dividers [Bromley-Brits 2011]; keep the PC out of sight of the pool [HVS]. Ceiling curtains on tracks let you hide the room for cued trials [Vorhees & Williams 2024]. Check access routes before ordering a large pool [HVS].

Going deeper

Morris showed that enclosing the pool in curtains dropped performance to chance, confirming that distal room cues drive place learning [Morris 1984]. Report the set-up under ARRIVE item 9 [Percie du Sert 2020].

Sources: Bromley-Brits et al. (2011). Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice. Journal of Visualized Experiments (53). doi:10.3791/2920; doi:10.1016/j.crtox.2024.100151; gov.uk/government/publications/care-and-accommodation-of-animals-in-science-advisory-standards; Morris (1984). Developments of a water-maze procedure for studying spatial learning in the rat. Journal of Neuroscience Methods 11(1):47-60. doi:10.1016/0165-0270(84)90007-4; doi:10.1371/journal.pbio.3000410

How should I set up the room, pool and platform for the water maze?

Camera above the pool centre, pool nearly filling the image, distal cues far away, diffuse lighting. 1) Pool about 1.8–2 m diameter with 30 cm of wall above about 30 cm of water. 2) Platform just below the surface, its top 1/50 to 1/100 of the water area. 3) Four uplighters low enough not to be seen from the water. 4) Tracking PC out of sight of the pool.

More detail

Room ideally square or ringed by screens; bigger rooms help with distal cues. Pool stand at a comfortable height on locking castors (a full pool can exceed 1.5 t, and movement changes calibration); pool truly circular with a uniform interior (black or white for contrast). Put subjects in gently facing the poolside and start the trial as they enter. HVS advises water about 13 °C below body temperature. Mice often use a 10 cm platform in a smaller pool; in a large pool use a larger platform (e.g. about 20 cm). Small pools can be shallower. Check access routes for large pools.

Going deeper

Pool size, platform size and water temperature depend on species and protocol, so ask HVS if you are unsure. Camera mounts over 1 m or wide-angle lenses suit low ceilings.

What noise should I watch out for in the testing room?

Keep the room quiet and avoid sudden noises. Rodents hear ultrasound that we cannot, so fix dripping taps, keep squeaky trolleys out during trials and keep monitors away from the apparatus. Keep people traffic to a minimum. Your local ethics committee, licence conditions and vet always take precedence.

More detail

UK guidance lists dripping taps, trolley wheels and computer monitors as sources of ultrasound (above 20 kHz), and warns against sudden, irregular noise [Home Office]. Better environmental control reduces variability, which can reduce the number of animals needed [Home Office].

Going deeper

An ultrasound detector helps you find hidden sources. A noise on one side of the room can also act as a directional cue, so keep sound sources the same on every side of the room and from day to day.

Sources: gov.uk/government/publications/care-and-accommodation-of-animals-in-science-advisory-standards

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.

More detail

Water maze and open field protocols use at least 30 minutes of room acclimation [Bromley-Brits 2011; Seibenhener & Wooten 2015]. Acclimatisation after transport is set by national rules or a competent person [Home Office]. ARRIVE asks you to report acclimatisation [Percie du Sert 2020].

Going deeper

Keep the settling time the same for every animal and every day, and record it. We found no source for a best time of day: keep it consistent and report it.

Sources: Bromley-Brits et al. (2011). Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice. Journal of Visualized Experiments (53). doi:10.3791/2920; Seibenhener & Wooten (2015). Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice. Journal of Visualized Experiments (96). doi:10.3791/52434; gov.uk/government/publications/care-and-accommodation-of-animals-in-science-advisory-standards; doi:10.1371/journal.pbio.3000410

What should I clean the maze or arena with between animals?

Clean between animals to remove urine, faeces and scent trails, and let the surface dry fully before the next animal. Ethanol is common; some labs use hypochlorous water because it leaves less smell. Use the same cleaner throughout a study. Your local ethics committee, licence conditions and vet always take precedence.

More detail

Protocols differ: 95% ethanol for an open field [Seibenhener & Wooten 2015], 10–75% ethanol on Barnes tables [Gawel 2019], or hypochlorous water [Komada 2008]. Ethanol vapour can affect behaviour, so ventilate and let it evaporate [Gawel 2019]. In the water maze, remove faeces and change the water regularly [Vorhees & Williams 2024].

Going deeper

Mixed practice: no cleaner is best for all tests. Rotating the Barnes table as well as wiping it stops odour trails being used as cues [Gawel 2019]. Check flammable solvents with your safety team.

Sources: Seibenhener & Wooten (2015). Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice. Journal of Visualized Experiments (96). doi:10.3791/52434; Gawel et al. (2019). Assessment of spatial learning and memory in the Barnes maze task in rodents—methodological consideration. Naunyn-Schmiedeberg's Archives of Pharmacology 392(1):1-18. doi:10.1007/s00210-018-1589-y; doi:10.3791/1088; doi:10.1016/j.crtox.2024.100151

What lab conditions should I record and report?

You should record and report every condition that could affect behaviour: room and water temperature, light level (lux) and light source type, time of day, who ran the animals, and any unusual events. These go into your methods. Your local ethics committee, licence conditions and vet always take precedence.

More detail

ARRIVE 2.0 asks for when, where and how often procedures were done, acclimatisation, randomisation, blinding and humane endpoints [Percie du Sert 2020]. Record water temperature daily [Vorhees & Williams 2024]. Report the light source type as well as lux, since lux is weighted to human vision [Peirson 2018].

Going deeper

Randomisation is reported in only about 30–40% of papers and blinding in about 20% [Percie du Sert 2020], so reporting them clearly strengthens your work.

Sources: doi:10.1371/journal.pbio.3000410; doi:10.1016/j.crtox.2024.100151; doi:10.1016/j.jneumeth.2017.04.007

How can staff see to work without disturbing the animals or tracking?

Use a shaded desk lamp at the screened-off PC, aimed away from the maze, and avoid sudden changes in light levels. Your local ethics committee, licence conditions and vet always take precedence.

More detail

Use dim, indirect working light placed so that it neither shines on the pool nor shows on the camera image. Lighting should be sufficient and glare-free, avoiding large differences in illuminance [HSE HSG38; HSE lighting]. A dimmable lamp lets you set the minimum light needed.

Going deeper

Our suggested practice: check the lamp does not show on the camera image.

Sources: hse.gov.uk/pubns/books/hsg38.htm; hse.gov.uk/humanfactors/topics/lighting.htm

For the whole set-up in one place, see Morris water maze (MWM) apparatus.

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