A Protocol to Assess Time-of-Day-Dependent Learning and Memory in Mice Using the Novel Object Recognition Test

Abstract

Changes in learning and memory are important behavioral readouts of brain function across multiple species. In mice, a multitude of behavioral tasks exist to study learning and memory, including those influenced by extrinsic and intrinsic forces such as stress (e.g., escape from danger, hunger, or thirst) or natural curiosity and exploratory drive. The novel object recognition (NOR) test is a widely used behavioral paradigm to study memory and learning under various conditions, including age, sex, motivational state, and neural circuit dynamics. Although mice are nocturnal, many behavioral tests are performed during their inactive period (light phase, subjective night) for the convenience of the diurnal experimenters. However, learning and memory are strongly associated with the animal's sleep-wake and circadian cycles, stressing the need to test these behaviors during the animals' active period (dark phase, subjective day). Here, we develop a protocol to perform the NOR task during both light (subjective night) and dark (subjective day) phases in adult mice (4 months old) and provide a flexible framework to test the learning and memory components of this task at distinct times of day and associated activity periods. We also highlight methodological details critical for obtaining the expected behavioral responses. Key features • Enables analysis of learning and memory in mice during both active (dark) and inactive (light) phases. • Allows for switching the time-of-day-dependent familiarization and recognition to study the impact of activity and sleep-wake cycle on cognitive performance. • Details the environmental and experimenter-dependent conditions that can impact behavioral responses. • Provides a flexible, adjustable platform for testing variable experimental conditions such as age, sex, learning and memory components, and genetic manipulations.

Keywords: Behavioral assay; Circadian rhythms/time of day; Learning and memory; Mice behavior protocol; Novel object recognition.

Figure 1.. Novel object recognition (NOR) test setup.

(A) Setup for behavioral testing, including the testing arena (1) shown with two different objects (2–3) placed a minimum of 5 cm away from the arena wall (yellow dashed line). The location of the objects remains constant throughout experimental days. A camera (4) is mounted above the arena (5–6: camera holders) at a distance of 96.5 cm from the arena floor (red dashed line). Camera height can be adjusted as needed, depending on camera type and setup, as long as the entire arena is within the field of view. Illuminators for both daytime (7, visible light) and nighttime (8, infrared light) are arranged as shown, avoiding direct illumination of the arena (especially important for visible light; see General notes and troubleshooting). The black dashed line corresponds to the arena width, 45 cm. (B) The different objects used for the NOR test in this protocol. On day 2 of the experiment, two identical objects are placed in the arena. One of the objects is replaced with a novel/different object on day 3 (as shown in A). Scale bar = 10 cm.

Figure 2.. Example images of object exploration.

See also Videos 1–4. Still images extracted from videos 1–4 showing a mouse within the novel object recognition (NOR) arena exploring objects on day 2 (left panels) and day 3 (right panels) during the light phase (AM; top panels) and dark phase (PM; bottom panels) as labeled. Blue and yellow banners indicate detection of an exploration bout by BehaviorDepot (see Data analysis section). During day 2 (familiarization), two identical objects are present in the arena, while during the testing session (day 3), one of the objects is replaced with a novel object (object 1, yellow banner).

Video 1.. Light phase (AM)_Day2

Video 2.. Light phase (AM)_Day3

Video 3.. Dark phase (PM)_Day2

Video 4.. Dark phase (PM)_Day3

Figure 3.. BehaviorDEPOT used for the analysis of object exploration.

A screenshot of BehaviorDEPOT graphical user interface containing the steps (numbered 1–8) needed to perform exploration analysis.

Figure 4.. Validation of the novel object recognition (NOR) protocol.

(A) Quantification of object exploration, represented as the discrimination index (DI) of mice tested during the light (yellow boxes) and dark (gray boxes) phases, as labeled. DI is higher on day 3 compared to day 2, suggesting increased exploration of the novel object over the familiar object during both activity phases. (B) Quantification of object exploration represented as total exploration time for each object (familiar objects, blue; novel object, red) in mice tested during the light (yellow background) and dark (gray background) phases. Exploration time is similar for objects 1 and 2 on day 2 (familiar objects), while exploration time is lower for the familiar object (object 2; blue box) on day 3 compared to the novel object (object 1; red box). (C–D) Kinematics parameters, including total distance traveled (C) and velocity (D), in mice tested during the light (yellow) and dark (gray) phases. No difference between the parameters is observed. N = 8 mice light phase; N = 8 mice dark phase. In A–B, data is shown as a box with whiskers (min-max), and the line is median. In C–D, data is shown as mean ± SEM. Data points represent individual mice. Statistical analysis by t-test; in B, exploration time was compared between objects within each day by a paired t-test. P value on the plots, ns = non-significant (P > 0.05). See also Figure S1.