Simultaneous assessment of cognitive function, circadian rhythm, and spontaneous activity in aging mice

Abstract

Cognitive function declines substantially with age in both humans and animal models. In humans, this decline is associated with decreases in independence and quality of life. Although the methodology for analysis of cognitive function in human models is relatively well established, similar analyses in animal models have many technical issues (e.g., unintended experimenter bias, motivational issues, stress, and testing during the light phase of the light dark cycle) that limit interpretation of the results. These caveats, and others, potentially bias the interpretation of studies in rodents and prevent the application of current tests of learning and memory as part of an overall healthspan assessment in rodent models of aging. The goal of this study was to establish the methodology to assess cognitive function in aging animals that addresses many of these concerns. Here, we use a food reward-based discrimination procedure with minimal stress in C57Bl/6J male mice at 6, 21, and 27 months of age, followed by a reversal task to assess behavioral flexibility. Importantly, the procedures minimize issues related to between-experimenter confounds and are conducted during both the dark and light phases of the light dark cycle in a home-cage setting. During cognitive testing, we were able to assess multiple measures of spontaneous movement and diurnal activity in young and aged mice including, distance moved, velocity, and acceleration over a 90-h period. Both initial discrimination and reversal learning significantly decreased with age and, similar to rats and humans, not all old mice demonstrated impairments in learning with age. These results permitted classification of animals based on their cognitive status. Analysis of movement parameters indicated decreases in distance moved as well as velocity and acceleration with increasing age. Based on these data, we developed preliminary models indicating, as in humans, a close relationship exists between age-related movement parameters and cognitive ability. Our results provide a reliable method for assessing cognitive performance with minimal stress and simultaneously provide key information on movement and diurnal activity. These methods represent a novel approach to developing non-invasive healthspan measures in rodent models that allow standardization across laboratories.

Keywords: Aging; Automated home-cage; Behaviour; Phenotyper; Spatial memory.

Fig. 1

Depiction of the Ethovision Phenotyper used for analysis of the effects of aging on spontaneous movement and cognitive function. The cages were transparent plastic (L  =  30 × W  =  30 × H  =  35 cm) and bedding was made of cellulose free paper. Water was available ad libitum and a shelter with nesting material was available for sleeping. The activity of the animals was continuously recorded for 90 h using EthoVision tracking software. An infrared-sensitive video camera above the arena recorded all movements and data were sampled at a rate of 15 fps and smoothed using EthoVision software. Immediately prior to initiation of tracking, a pellet dispenser and a CognitionWall were inserted into the PhenoTyper. The CognitionWall has three entrances (left (L), middle (M), and right (R)) placed in front of a food dispenser. During the initial discrimination phase of the study, the animals were required to enter the CognitionWall through the left entrance and were rewarded with a food pellet using FR5 schedule. The criteria for success were 80% and calculated based on the success rate of the trailing 30 entries. After 48 h, the correct entrance was reversed to the right entrance and the study terminated at 90 h. Data were uploaded to AHCODA-DB (mousedata.sylics.com) for analysis and independently validated using R

Fig. 2

Data derived from tracking animal movements during the 90 h experimental period. Analyses included a total distance moved, b time spent moving, c number of moving segments, and d acceleration. Data are averaged over hourly intervals during both the light and dark phases of the L:D cycle. Dark horizontal lines represent the dark phase of the cycle. Data represent mean + SEM calculated for hourly intervals

Fig. 3

Summary of spontaneous movement parameters during the light and dark phases. Each movement parameter was averaged over the light and dark phases over the entire 90 h study. a Total distance moved, b total time spent moving (seconds), c number of moving segments, and d acceleration (cm/s²). Data represent mean + SEM for 9–11 animals/age group. *p < 0.05

Fig. 4

Percentage of mice of each age group that reached criteria during the initial discrimination task. Success rates were calculated as the percentage of correct entries using a moving window based on the last 30 entries into the CognitionWall. Data indicate that 100% of 6-, 21-, and 27-month-old animals reach 73% correct responses but the percent of 21- and 27-month-old animals reaching higher success rates is reduced. At a success rate of 83%, differences were evident between 6 and 21 and 27-month-old animals (*p < 0.05). The 80% success rate was chosen for all subsequent comparisons, as in previous publications

Fig. 5

a Entries required to reach 80% success rate for each age group during the initial discrimination phase of the study. Each “step” in the curve represents a mouse that reached the learning criterion at the respective number of entries. Log-rank test for differences between two or more Kaplan–Meier survival curves indicates that the cohort of 21- and 27-month-old animals requires more entries to reach the 80% success rate compared with the 6-month-old animals, whereas there was no statistically significant difference between 21- and 27-month animals. b Entries required to reach 80% success rate for each age group during the reversal phase of the study. Data indicate that the cohort of 21- and 27-month-old animals is severely impaired in this task and less than 35% of animals were successful compared to 75% for 6-month-old animals

Fig. 6

Number of errors (incorrect entries) to reach 80% during the initial discrimination (a) and the reversal (b) phases of the task. Incorrect entries tended to increase with age in the initial discrimination phase. During the reversal, the number of errors increased at 21 months and then declined at 27 months of age. Further analysis indicated that the decline in errors at 27 months of age was due to a reduction in the number of old animals that reached criteria and therefore are not included in the group (refer to Fig. 7)

Fig. 7

Entries required to reach 80% success rate for each age group during the initial discrimination (a) and reversal (b) phases of the study. Each point represents the number of entries required for an individual animal to reach the 80% success rate. Note that 21- and 27-month-old animals separate into two groups—those with performance similar to young animals and those with performance that is impaired compared to young animals. All of the impaired animals identified during the initial discrimination phase of the study at 27 months of age were not able to reach the 80% criteria in the reversal phase of the study. Mice that did not reach criteria are identified in the graph with arbitrary values above 5000 entries (separated by the dashed line)