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Behavioral Test Questions

What should the water temperature be (if water is used in the apparatus)?

By June 24, 2019October 18th, 2019No Comments

Water-based behavioral tasks, like any environmentally specific experiment, are influenced by such factors as light and temperature. Given that experimental subjects, mice and rats, are submerged in water for such assays as the forced swim test (FST) and Morris water maze (MWM), it is no surprise that the temperature of the water influences their performance. Both of these tasks are predicated on the idea that rodents find water to be aversive and are therefore motivated to escape (in the MWM) or exhibit coping strategies to endure the unpleasant situation (in the FST). Researchers must consider what temperature is associated with this aversive condition and how to modulate that in order to alter performance in these tasks.

Morris water maze

The temperature of the MWM is normally set to be approximately 13°C lower than the body temperature of the animal. This is considered sufficient to induce anxiety and therefore motivate the animal to escape by finding the platform.

In the MWM, a decrease in temperature is often associated with faster platform finding. This is surmised to be a result of cold water being an aversive stimulus; while the MWM is already considered to be environmentally aversive, the colder water increases this factor and encourages the animals to more quickly seek an escape. For instance, a decrease from 24°C to 19°C has been shown to significantly decrease the latency to find the platform [1] in mice. This finding has been repeated many times and is associated with increased levels of the stress hormone, corticosterone. Adult, male rats tested in the MWM at 19°C exhibit significantly higher levels of corticosterone than rats tested at 25°C, for instance.[2]

This relationship between temperature and performance is not necessarily limitless, however. Performance is compromised at extremely low temperatures which may impact cognition, such as 12°C. [3]

Forced swim test

For the forced swim test, the water used is normally at room temperature (23°C to 30°C[4][5]). Temperatures above this range may not be aversive enough to induce an immobility response, as seen with mice tested at 35°C who do not develop this behavior.[6] On the contrary, temperatures lower than this may actually decrease learned immobility in rats, as has been seen when rats are tested in the FST at 20°C. [7] Furthermore, colder temperatures during prolonged FST sessions causes a significant drop in brain temperature, which is in turn associated with decreased cognitive performance. When tested in the FST for 10 minutes at 25°C, rats exhibit significant brain hypothermia.[8] This same duration of test in warm water (37°C) does not cause this effect, however. This is important since it is known that such decreases in brain temperature inhibit neuronal activity. Thus, the immobility seen in prolonged FST sessions may be misinterpreted as a form of learned helplessness when in fact it is actually a neurological deficit.


As is often the case, behavioral differences exist between strains of rodents regarding the parameter of water temperature in such tasks as the FST. For instance, researchers in Switzerland tested three different water temperatures (20°C, 25°C and 30°C) on performance in the FST in two different commonly used strains of mice, specifically the C57BL/6J and BALB/c strains.[9] As the water temperature was increased, the two strains showed opposite reactions: the C57BL/6J mice floated for longer, while the BALB/c mice floated for less time. Interestingly, both strains showed decreased levels of the stress hormone, corticosterone, in response to increasing temperatures. This suggests that the colder temperatures are more aversive than warmer temperatures, but that coping strategies with this unpleasant condition differ between the two strains.


Of important note, is that differences in temperature responding in these tasks may exist between male and female subjects. In one example of such a difference, female rats were tested at temperatures similar to those discussed above (25°C and 19°C) with no difference found between these conditions.[10] This may be due to complications of testing female rats at different points in the estrus cycle. Previously, it has been shown that at different points in the estrus cycle, rats react differently to temperatures in the MWM. Specifically, female rats during proestrus tend to perform better in warm (33°C) water while rats during estrus perform better in cold (19°C) water.[11]


Clearly, performance in the MWM and FST are influenced by water temperature. In most cases, it appears that decreased temperatures in the MWM are associated with decreased latency to find the platform.

It is important to note that, even with temperatures of water that fall within the normal range discussed in this article, rodents subjected to repeated water tests are susceptible to hypothermia. In one study, repeated 45-second trials in the MWM at 20°C with 30-second inter-trial intervals was associated with a 9°C temperature drop in the tested mice.[12] This can be somewhat alleviated with higher water temperatures (such as 35°C in the FST),[13] or with increased inter-trial intervals. However, researchers should be careful to monitor the core temperature of their subjects in order to avoid such a hypothermic effect.


  1. Stasko, M. R., & Costa, A. C. . (2004). Experimental parameters affecting the Morris water maze performance of a mouse model of Down syndrome. Behavioural Brain Research, 154(1), 1–17.
  2. Sandi, C., Loscertales, M., & Guaza, C. (1997). Experience-dependent facilitating effect of corticosterone on spatial memory formation in the water maze. The European Journal of Neuroscience, 9(4), 637–642.
  3. Morris, R. G. M. (2008). Morris water maze. Scholarpedia, 3(8), 6315.
  4. Can, A., Dao, D. T., Arad, M., Terrillion, C. E., Piantadosi, S. C., & Gould, T. D. (2012). The Mouse Forced Swim Test. Journal of Visualized Experiments : JoVE, (59).
  5. National Research Council (US) Committee on Guidelines for the Use of Animals in Neuroscience and Behavioral Research. (2003). Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. In The National Academies Collection: Reports Funded by National Institutes of Health.
  6. Arai, I., Tsuyuki, Y., Shiomoto, H., Satoh, M., & Otomo, S. (2000). Decreased body temperature dependent appearance of behavioral despair in the forced swimming test in mice. Pharmacological Research, 42(2), 171–176.
  7. Jefferys, D., & Funder, J. (1994). The effect of water temperature on immobility in the forced swimming test in rats. European Journal of Pharmacology, 253(1–2), 91–94.
  8. Taltavull, J. F., Chefer, V. I., Shippenberg, T. S., & Kiyatkin, E. A. (2003). Severe brain hypothermia as a factor underlying behavioral immobility during cold-water forced swim. Brain Research, 975(1–2), 244–247.
  9. Bächli, H., Steiner, M. A., Habersetzer, U., & Wotjak, C. T. (2008). Increased water temperature renders single-housed C57BL/6J mice susceptible to antidepressant treatment in the forced swim test. Behavioural Brain Research, 187(1), 67–71.
  10. Anderson, E. M., Moenk, M. D., Barbaro, L., Clarke, D. A., & Matuszewich, L. (2013). Effects of pretraining and water temperature on female rats’ performance in the Morris water maze. Physiology & Behavior, 122, 216–221.
  11. Rubinow, M. J., Arseneau, L. M., Beverly, J. L., & Juraska, J. M. (2004). Effect of the estrous cycle on water maze acquisition depends on the temperature of the water. Behavioral Neuroscience, 118(4), 863–868.
  12. Iivonen, H., Nurminen, L., Harri, M., Tanila, H., & Puoliväli, J. (2003). Hypothermia in mice tested in Morris water maze. Behavioural Brain Research, 141(2), 207–213.
  13. Arai, I., Tsuyuki, Y., Shiomoto, H., Satoh, M., & Otomo, S. (2000). Decreased body temperature dependent appearance of behavioral despair in the forced swimming test in mice. Pharmacological Research, 42(2), 171–176.
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