Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Abstract

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Figure 1:. Behavioral testing preparation.

(A) Open-field-testing arena assembly with part A corresponding to the inner wall, part B as the outer wall, and part C as the base. The finished arena will have two outer walls (parts B) that run the entire edge of the base and two inner walls (parts A) that fit between the outer walls on the adjoining edges of the base (part C). All walls will rest on top of the base. (B) Representative arrangement of arenas on a 0.62 m high table, 60 × 90 cm environmental cues, lights, and a camera for a testing area that allows the capture of all four arenas simultaneously. (C) A curtain hides the experimenter and computer system from mice during trials. The overhead lights are on for the purposes of taking this photograph, but during testing, only the floor lamps are on. Also, one of the environmental cues has been removed for this photograph of the testing area, but during testing, there is a fourth cue in front of the arenas, facing the all black cue behind the table. (D) A representative object (and ruler for scale) that is appropriate for OLT or NORT testing with mice.

Figure 2:. Arena configuration for trials.

(A) Open-field testing arena without objects for habituation session. The black arrow indicates a release corner. This corner should be the same relative location in each arena and be consistent for every mouse being tested and for every trial. (B) For the training trial, two different objects are secured to the open field at 6 × 6 cm away from their respective walls. (C) For the OLT, one object is moved to a new location, also 6 × 6 cm away from the walls and not the release corner. (D) For the NORT, the object that was stationary in the OLT is replaced with a novel object while the moved object from the OLT is now the familiar object.

Figure 3:. Behavioral testing data with wild type adult C57Bl/6 mice.

(A) Comparison of percent total investigation time of the moved object during the training trial versus the OLT shows significant increase in investigation, after the object moved. ****p < 0.0001, paired t-test. (B) Representative results for moved object investigation during training and OLT trials displayed as a discrimination index similarly show a significant increase in investigation of the object after it is moved. ****p < 0.0001, paired t-test. (C) Percent total investigation time of the novel object in the NORT shows significant preference for investigating the novel object. **p = 0.0024, one-sample t-test vs. 50%. (D) Representative results for novel object investigation in the NORT displayed as a discrimination index similarly show preference for investigating the novel object. **p = 0.0024, one-sample t-test vs 0. (E) Representative results of a NORT analysis involving two different groups of mice. Group B differs significantly from 50% by one-sample t-test (**p = 0.0024), but group A does not (p = 0.5837). In a separate analysis, to compare groups, a two-sample Mann-Whitney test is used because of the uneven group sizes and no significant difference in investigation is found (p = 0.66, ns). (F) Percent time with an object during validation trials in a small sample size shows a trend towards aversion to the object. p = 0.2159, one-sample t-test. (G) With a larger sample size and the object from (F) used as a novel object in a NORT, a significant aversion to the object is found, even though it is the novel object. *p = 0.0270, one-sample t-test. This is an example of a technical failure in object selection. Data are presented as mean ± SEM. Data from panels C-E are adapted from a previous publication.