One thing to consider for test battery order is the impact of stress on the animal for each task, which may come from innate aversiveness (bright lights, swim stress) or task difficulty. We know that stress impacts learning and cognition, and if this might complicate your variables of interest perhaps starting with the least stressful to most stressful would be a way to go. For example, unless you are using very bright lighting, open field exploration then NOR you might begin with, and maybe MWM last. Counterbalancing test order could potentially control for test order effects but might add unwanted variability in performance for each.

Anantha, others had exactly the same problem. So they "defined" it: https://www.mousephenotype.org/impress

Hi, I would suggest you follow:

1. NOR 2. Y maze 3. Marble burying 4. Open field assay 5. Light/dark box 6. Morris Water maze. Also, are you planning on these studies to test phenotype in your genetically modified animals? I ask this because it would be better to use some animals exclusively for a few of these studies and then use the rest for the rest. Ideally, I would go for studies involving a little stress such as dark/light and MWM with separate set of animals altogether to avoid any confounding factors..

Hi

By seeing your list of apparatus you have it seems there are two sets of behavioural phenotypes you want to characterize

1. Anxiety [ Y maze, Open field assay, Light/dark box]

2. Learning & memory [NOR, Marble burying, Morris Water Maze]

If you have option of having more animals, in my suggestion you can characterize these two separately with different sets of animals. In case, your animals are not enough, very precious (transgenic/knockout or something) then you have to be careful in ordering them in such a manner that the swim stress while training for MWM should not hamper the training for other tests like familiar object memory formation in NOR. Hope you got the point. Anyways MWM should be the last.

Best wishes for your experiments.

As a rule, tests for anxiety-like behavior should always be performed first, since they can be greatly affected by any previous experience. So I would suggest:

1. Light/Dark box

2. Open field

3. Marble burying

4. Novel object recognition

5. Y-maze (depending on how you are conducting the maze/what you are analyzing)

6. Morris water maze (probably the most stressful)

Good luck!

Dear Anantha,

From my experience, it is often difficult to exploit all behavioral data from animals successively evaluated in a battery of tasks. This is all the more true when, as you plan, rodents are going to be tested in a high number of paradigms pertaining to different domains (activity, anxiety/depression, learning and memory).

Therefore, I would first advise you, if possible, to review your initial study design so that you can separately investigate learning & memory (1. Y Maze 2. NOR 3. Water Maze) and other functions (1. Open Field 2. Marble Burying Test 3. Light/Dark Box) with distinct batches of animals.

If for some reason this is not a viable option, I would propose the following order for your behavioral testing: 1. Open Field (provided that you measure activity levels rather than anxiety) 2. Y Maze 3. NOR 4. Marble Burying Test 5. Water Maze. Although the two last assays certainly increase stress in mice, I consider that the Water Maze has a higher aversive component because the associated training period lasts longer. As for the Light/Dark Box, I would abandon the idea to include it.

I agree with comments that advise to test different functions with different sets of animals. It would really be ideal to use one set of animals to assess learning and memory (1. Y maze 2. NOR 3. MWM) and another set for anxiety (1. Light/Dark Box 2. Open Field 3. Marble Burying). If this is not possible, you have to decide do you put the the anxiety tests at the beginning, which can be more effected by previous behaviors and make the order (1. Light/Dark Box 2. Open Field 3. Marble Burying 4. Y Maze 5. NOR 6. MWM) or put the most stressful tests at the end in which case I would alter the order to be (1. Open Field 2. Light/Dark Box 3. Y Maze 4. NOR 5. Marble Burying 6. MWM). Many of these tests have unique stressors and are probably hard to rank, but MWM is by far the most stressful. If you have enough mice and opt not to test in batches, you also could counter-balance the order the tests are received in, or save MWM for last and counter-balance the less aversive tests to potentially minimize effects of testing order. Good luck!

Thank you very much for the kind input Drs. Hoffman and Rajagopal. We had a discussion about it and your inputs helped us a lot in designing the experiment. Thank you Dr. Foerster for the link. I checked the link but could not find information I was looking for. Could you help me in reaching the specific location in the website? 

Drs. Chakravarthy, Delotterie and Falco, your suggestion that we will be better off using more number of animals for all these experiments as we are estimating two different behavioral patterns makes so much sense. We decided that we would follow this for all our upcoming experiments. 

We are using Y maze to determine spontaneous alteration in mice upon our treatment Dr. Anacker. Thank you very much for the input.

Yes Dr. Jadhav. We are planning to do biochemical/pharmacological studies post our behavioral experiments. Thank you very much.

Hi,

I do behavior on a regular basis and can say that you should do the ones that cause the most stress last, as already mentioned. However, and depending on the protocol and questions you intend to ask, you can repeat some behavioural tests to see the evolution of the behaviour during your protocol.

I would do Marble and NOR (same day), Light-dark and open field first (same day), in this sequence. then I would do Y-maze (memory) and eventually repeat the first four to account for differences. Last, would do the MWM and then repeat the first four to see an evolution (or lack thereof) of parameters.

Hope to have been helpful,

J

check my publications we have performed behavior expts in y-maze. PMID's

25609594,25017367,24123667,23575834. It may be useful

I am also of the opinion that you should redesign your study and use different sets of animals for anxiet, learning and memory as OFT can affect the behaviour of animals in light/dark test and vice versa. You can perform one test each of anxiety and learning /memory on one set of animal,  and another test of anxiety and learning /memory on another set of animal.  

Dear Anantha,

The exposition to each experimental apparatus may lead to a broad sort of secondary effects, which may impact the results of the following test. For example, the anxiogenic condition of the open field test and resulting neophobia may impact the result of a following marbles test. 

Unless you want to correlate the results of different tests, I suggest you do not not perform them all in a single animal, or at least not on the same day (if your intervention allows that). 

Thiis is a more complicated questions than can be answered in a post.  Feel free to email me and we can set up a phone conversation if you want help  [email protected]