I am planning on completing novel object recognition testing in C57 mice but I need to validate the objects in a separate cohort to ensure there is not innate preference to any object. Does anyone have a protocol for this as I cannot find any specific within the literature, just that it has been completed.
Thanks
Dear Jessica,
I understand your concerns and they are very valid, especially for an assay such as the NOR that absolutely relies on impartiality!
In my lab we first validate the objects by introducing them to a batch of mice in the classic NOR set-up (two different objects in the roaming area) and then analysing the time each mouse spends exploring each object in a 5 minute trial. We then plot the exploration time of Object A / Object B to come out with the bias ratio for each mouse in the batch. We will proceed to choose the two objects where this bias value is closest to 1.0, as these objects are deemed to be “equally attractive”. Anything from 0.8 to 1.2 should work well for your actual NOR study.
You could perform this validation phase with about 20 mice and it’s better if you could vary the strain, age, and sex of these mice to make sure the validation is precise.
Lastly, the objects you use should ideally vary in shape, size, colour, and texture. In my experience mice can get quite excited at times and may jump up on the object itself and sit there (rendering that particular trial useless) so it would be helpful to find something that is tall and not flat at the top.
Hope this helps!
Dear Sarivin,
Yes, this helps a lot thank you very much. We have had a couple of answers about this from members in our institution who suggest similar methadologies.
Dear Jessica,
before validating the objects you should validate the arena, in order to verify if there is a spatial bias (for example due to differences in lighting). You can divide the arena into four quadrants and verify that the percentage of exploration for each quadrant is not statistically different from chance level. Here you can find indications for the validation of the arena:
https://www.researchgate.net/publication/354131226_The_4-Hole-Board_Test_for_Assessment_of_Long-Term_Spatial_Memory_in_Mice
Subsequently, you should validate the objects. Expose naive animals to the two different objects together for 5 minutes and then verify that the percentage of exploration for each of the objects is not significantly different from chance level. Here you can find indications for the validation of the objects:
https://www.researchgate.net/publication/259956779_Behavioral_Methods_for_the_Study_of_the_Ras-ERK_Pathway_in_Memory_Formation_and_Consolidation_Passive_Avoidance_and_Novel_Object_Recognition_Tests
Best,
Raffaele