I'm having some trouble with my mice freezing when they are placed in the Morris water maze. I understand that this is indicative of anxiety, but I'm not sure how best to remove this element when conducting these experiments. I try to handle the mice daily for at least a week prior to commencing the experiment and the water is kept at around 28°C. Any suggestions would be greatly appreciated.
Do not try to remove the freezing behavior. It is an indication of e.g. strain differences. Measure it between strains or between conditions or medications alternatively switch to a dry maze. You find a model in my papers.
Do not try to remove the freezing behavior. It is an indication of e.g. strain differences. Measure it between strains or between conditions or medications alternatively switch to a dry maze. You find a model in my papers.
Completely agree. There are important strain differences and not all mice are good for the MWM. In addition, if the mice are well handled (as you said), this is purely anxiety and as you can see, it can interfere with learning in the MWM.
Freezing behavior is an adaptative response in nature. Thus, it is a normal defensive behavior that is evoked when the animals face dangerous stimuli, as it is the context of water-maze task. In your case, this response is not affected by handling since that the aversive stimuli is not you but the water-maze cues. I think that "removing" this behavior, if possible, will grandly affect the performance of your experimental subjects on the task. Put in mind that, in this context, freezing is an unconditioned response to the environmental demand. The best you can do is to cope with.
By freezing, do you mean 'floaters'? Besides what others have already answered, I would like to suggest that you may be making it too difficult for the mice in the initial stages of training. If the mice find pre-training in the MWM too difficult, they will 'learn' to float. You do not want floaters. A solution is to add days to the pretraining so the learning curve is less steep. Good luck.
Thank you everyone for your helpful replies! Roger, yes I'm referring to "floaters". I had thought that adding days prior to commencing the training (as you suggested) might help with this problem, so would you suggest that I do this in the absence of the escape platform and visual cues?
There are a few standard pretraining schedules. Most start with the swimming to a visible platform, either raised above the water or to a flag placed on top of the submerged platform. Mice float when they do not learn/forget that there is a scape platform or when the cost of floating is less than struggling to find the platform.
Also, do not pick up a floating mouse, guide the mouse to the plaform, then leave it there for 15 s or more.
I recommend you take a look at the methods section of
Daumas S, Sandin J, Chen KS, Kobayashi D, Tulloch J, Martin SJ, Games D, Morris RG. Faster forgetting contributes to impaired spatial memory in the PDAPP mouse: deficit in memory retrieval associated with increased sensitivity to interference? Learn Mem, 2008; 15: 625-32.
Also, you can keep the water at 25 C if your Animal Care protocol lets you.
Water temperature should be around 22-24 C for reduce freezing behavior of mice.
Why reduce freezing? It is an important behavior that you should analyze.
I agree that water temperature is very high. Maybe rats are just enjoying the warm water and the behavior is not freezing. Lower the temperature to 22-24 as Ilknur Dursun suggests.
The temperature of the water and the anxiety level/depression level of the animal, the strain and/or age of the mice all effect the floating times. I used C57Bl/6 male and female mice on that task before, as long as you are close to (or slightly lower) than room temperature you should be fine. You can make sure they receive adequate amount of handling 4-5 days prior to training to reduce the anxiety.stress levels, also you can consider increasing the ITI to give them a break in between trials.
Something else to consider might be environmental conditions - check for high frequency noise pollution and reduce light levels if possible.
Hi Paul,
I think your water temperature is too high. 23 +/- 1°C would be better for C57 mice. Floating time is informative, you should think about its potential meanings in relation with your problematic. Other options than latency exist to assess spatial learning in the Morris maze. You could consider the search strategies used by your mice for instance (see Garthe and Kempermann). It would allow you to eliminate bias like floating time on your measure of spatial learning.
I just saw a video about this on JoVE web site. The researcher uses "paddling" instead of swimming. It seemed to work well.
In my point of view, you should take a care and control the animal's anxiety level, the temperature of water and the experimental environnement. Make sure that there is no particular odor that can be disrupt the behavior of the animal. In deed, this later point was highlighted by Banik A and Anand A. (2011) in a paper entitled:''Loss of learning in mice when exposed to rat odor: a water maze study.''
Hi Paul
Floating (or freezing) can have several reasons:
Unless this was an experiment asked by a reviewer of your manuscript, I would also recommend to move to more natural tasks for mice, since the odds of seen a mouse swimming in nature are really low. Try Barnes maze, or even the paddling in shallow water task for more representative data.
http://www.jove.com/video/2608/shallow-water-paddling-variants-of-water-maze-tests-in-mice