Background

Behavioral testing in rodents enables evaluating multiple aspects of cognitive processes, including attention, learning, memory, spatial navigation, fear, and anxiety, under both normal and pathological conditions [1,2]. These tests vary in complexity and structure, with some exploiting animals’ natural instincts (such as escape from danger [3] or grooming [4]) and others employing complex training sessions to elicit learned responses (such as maze navigation [5] or pellet reaching [6]). The novel object recognition (NOR) test is routinely utilized to quantify learning and memory in mice, relying on their inherent drive to explore objects within their environment [1,7–9]. The test involves two sessions where a mouse freely explores an arena. Initially, the arena contains two identical objects for familiarization. In the second session, one object is replaced with a novel object. It is expected that the mouse's natural curiosity will lead it to spend more time exploring the novel object, providing a measure of its learning and memory. NOR is a powerful paradigm due to its high degree of flexibility and adaptation to various experimental questions, for example, to quantify short-term vs. long-term memory, or to examine how different environmental conditions (such as time of day) or genetic perturbations (such as gene knockouts) impact these cognitive processes [10,11]. Importantly, mouse behavioral performance may be influenced by stressors such as light and noise [12], underscoring the importance of carefully calibrating the testing parameters to enable reliable, measurable responses.

Cognitive function is strongly influenced by the light/dark cycle and circadian clock [13,14]. The circadian clock is an intrinsic rhythm-generating system consisting of transcriptional-translational feedback loops involving core clock genes, which maintain a 24-h periodic regulation of multiple aspects of physiology, including cognition [15–17]. Numerous studies across various species including flies, rodents, and humans have shown that learning and memory abilities vary by time of day [13,14,18–27], while circadian disruptions due to altered light/dark or feeding cycles (such as jet lag or clock gene dysregulation) result in impaired cognitive performance in several behavioral tasks [28–37], including the NOR test [10,11]. Importantly, the NOR test offers a unique advantage for studying clock-related cognitive function as it enables separate investigation of learning and memory components like consolidation and recall by varying the time of day of object familiarization and recognition. Since circadian disruptions are linked to cognitive disorders such as depression [38–40] and Alzheimer’s disease [41–43], designing robust experimental paradigms to study the underlying mechanisms is crucial for advancing our understanding of these disorders and for developing novel treatments.

Here, we describe a protocol for the NOR test that is modified [8,10,11,44] to quantify time-of-day-dependent learning and memory in mice. The test consists of three consecutive days (24 h interval) and includes habituation (day 1; no objects), familiarization (day 2; identical objects), and testing (day 3; novel object; see Graphical overview). Moreover, the habituation day serves as an “open-field test” [1] to assess time-of-day-dependent locomotion and general anxiety. This protocol is highly flexible and can be adapted to test the effects of light/dark cycle and circadian clock perturbations on learning as well as both short-term and long-term memory. This protocol also provides insights and troubleshooting suggestions to ensure robust and reproducible results.