Novel Object Recognition Test for the Investigation of Learning and Memory in Mice

The overall goal of this behavioral assay is to test various aspects of learning and memory in mice. This method can help answer numerous questions in the area of rodent cognition, from the basics of developing a deeper understanding, for certain aspects of learning in memory and mice, as well as more complex issues, such as cognitive impairment, including disease models, such as Alzheimer’s disease, to the exploration of potential cognitive enhancement. The main advantage of the ORT, is its simplicity, efficiency and flexibility and studying learning and memory in mice.

Demonstrating this procedure will be Andrei Jeltyi, a lab technician from our lab. Begin by selecting mole sized objects that can be easily discriminated by the animal but have a similar degree of complexity in features, such as, texture, shape or brightness to minimize any potential induced object preference that may bias the results. Additionally, to further reduce induced preferences, ensure that mice are able to climb on both objects or neither object.

The ability to climb on an object may increase interest in exploration. Then to minimize the stress of bright lighting, use defused low lighting with the center of the maze illuminated around 20 lucks. Acclimate mice to any new testing rooms for at least one hour prior to use each day and keep the temperature and humidity similar to regular housing conditions.

Note, the mice should be handled one to two times a day for at least one minute, over the course of one to two weeks prior to testing. Lastly, for the main arena, use a square chamber made from white or black non-porous plastic contrasted to the color of the mouse. Begin by removing the mouse from its home cage and placing it in the middle of the open empty arena.

Allow for free exploration of the arena for five minutes. Once the home cage is empty, save it for use as a holding cage the next day. At the end of five minutes, remove the mouse and place it in a holding cage.

Do not return the mouse to its original home cage, or this may affect the behavior of the remaining mice to be tested. Then thoroughly clean the apparatus between mice, using 70%volume per volume ethanol. Next, begin the training phase or T1, by placing two identical objects in opposite quadrons of the arena.

For example, one in the north west corner and one in the south east corner. Fasten any objects that are too light to the floor with removal mounting putty. Counter balance the use of each set of objects as well as the location of the novel object to each of the four corners of the arena.

Be sure to know, which diagonal is used for which animal, so that the diagonal use on the training day is the shape as that used on the testing day for each mouse. 24 hours after habituation, remove the mouse from its home cage and place it in the center of the arena, equidistant from the two identical objects to begin training. Allow free exploration for a minimum of five minutes, if fusing a strain of mice that are known to have low locomotor or exploration activity, extend the trial to 10 minutes for all mice in the cohorts.

At the end of training trial, remove the mouse and place it in the holding cage. Once the home cage is empty, save it for use as the holding cage on testing day. Be sure to clean the apparatus and objects between mice.

Next, start the testing phase or T2, by placing the familiar object used during training and one novel object in opposite quadrons of the arena. Use the same locations as you use during training for each mouse. At the T1 to T2 interval of choosing, remove the mouse from its home cage and place it in the center arena, equidistant from the familiar object and the novel object.

Allow free exploration for 10 minutes. Finally, at the end of the testing trial, remove the mouse and place it in the holding cage. For analysis, exploration is defined as the nose directed towards the object within two to three centimeters with active vibracy sweeping.

However, the mouse sitting on the object, is not counted towards exploration time. For both T1 and T2, score the first five minutes and calculate the total exploration time for both objects for each session. If the mouse does not meet the minimum exploration time of 20 seconds for both objects, continue scoring past five minutes until total exploration exceeds 20 seconds.

Extend T1 and T2 time to 10 minutes for strains of mice that have low exploration and do not meet this minimum criterion by five minutes as observed during pilot testing. Finally, if mice do not reach a 20 second minimum of exploration for both objects for either T1 or T2 at 10 minutes, exclude them from the analysis as it cannot be confirmed that the animal spent enough time exploring to learn and discriminate. The object recognition task is an efficient and flexible method for studying learning and memory in mice, in which phosphodiesterase 2 inhibitors have been shown to improve during this task.

As compared to vehicle, administration of the PDE2 inhibitors bays 60-7550 or ND7001, significantly enhanced memory in a dose dependent manner, when given 30 minutes prior to training. When administered at different time points, relative to training and testing, the PDE2 inhibitor bay 60-7550 significantly enhanced his memory when given 30 minutes prior to training, as well as immediately after training and 30 minutes prior to recall. Once mastered, this technique can be completed in two to three days if it’s performed properly.

While attempting this procedure, it’s important to remember to run pilot studies and carefully choose the T1 to T2 interval, as choosing a wrong interval may result in a misaffect. Following this procedure, other methods like the Barn’s maze can be performed in order to answer additional questions like differences in learning, which cannot be fully addressed using the object recognition tests. After its development, this technique paved the way for researchers in the field of learning and memory to explore the basic biological mechanisms of memory, as well as explore potential mechanisms of various cognitive deficits in rodents.

After watching this video, you should have a good understanding of how to set up and design an appropriate experiment using the object recognition test, including running pilot studies, choosing objects and selecting the appropriate T1 to T2 interval. Thanks for watching and good luck with your experiments.