The duration of the EPM test could be either 5 min or 10 min. These behavioral parameters should be monitored: open-arm time/total time rate; the number of open arm entries/total entries rate; total motor activity: the total number of crosses between any two arms.

The entry in any arm is counted when all four legs of the mouse are crossed the entrance line of the arm.

Check the Nature Protocol article regarding this method:

https://www.nature.com/nprot/journal/v2/n2/full/nprot.2007.44.html

We also have published studies about EPM measurements in mice.

It is not an accepted approach to section or divide the open arm, e.g. distal ends or ends closer to the junction.

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Dear Akihiro, although the classical paradigm generally considers 5 or 10 minutes as duration and whole-arm entries, it can be interesting to perform a finer analysis to detect subtle phenotypical differences. Avoidance of the distal ends during the first 150 seconds could represent a mild anxious phenotype, which is easily overcome by your mice through few minutes of familiarization with the apparatus. I suggest evaluating these mice in other anxiety tests (as open-field and light-dark box) in order to better understand their phenotype.

 Dear Akhiro,

Indeed, classically the EPM lasts for 5- or 10 min. But there is of course no golden rule that you HAVE to make it last that long if you have good reasons to do otherwise (and you may have those). In addition, if the distal one-third of the open arm represents something meaningful it would be silly not to adjust. Scientific methods can always be improved.

However, one important word of caution. You ran the test for the 10 min initially while not 'cutting up' your open arms. So when you re-analyzed your data post-hoc you were not unbiased -anymore- as you had a 'hunch' animals would be different the first 150s and that there could be something up with the distal end of your open arms. Though I think your re-analysis is very interesting, it may have confounded your outcome. So now you have a testable hypothesis, meaning that you have to run another group of MiceX again, now unbiased, to see if the phenotype persists. 

Good luck!

Michael

Thank you all for your replies, and I'm sorry for the so long delay in my reply.

After my previous posting, I found a couple of papers. One is about the time course of exploratory behaviors, and the other about the duration at distal ends of open arms.

https://www.ncbi.nlm.nih.gov/pubmed/16084592

https://www.ncbi.nlm.nih.gov/pubmed/26241172

Based on these studies, it would be possible to interpret the data as suggesting that the mice X have slight anxious phenotypes.

I would like to confirm these results with new groups and other anxiety tests:)

But I have some further questions...

1) What is the elemental difference of anxiety expressed in EPM, light/dark test and open-field test?

2) Are there differences in neuronal structures engaged in these tests?

3) Between these tests, which one is the most sensitive?

It would be greatly appreciated if you could answer these questions.

Best regards,

Akihiro

Dear Ahikiro, these behavioural tests measure different types of anxiety. EPM measures acrophobia-induced anxiety (fear of height), light/dark test evaluates photophobia-induced anxiety (repulsion for light) and open-field test evaluates agoraphobia-induced anxiety (fear of open spaces). Since rodents are natural ly acrophobic, photophobic and thigmotactic (prefer moving along borders avoiding open spaces when exposed to a new environment), these tests are good models to study anxiety in mice and rats. As these tests focus on different kinds of anxiety, they can dissociate. You may find a pathological phenotype in one of the tests but not in the others.