All methods described here have been approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR), the University of Hong Kong.
1. General protocol
NOTE: This section is based on Deacon19.
2. BehavioralTest Protocol
This behavioral test battery was designed for the comprehensive and valid behavioral analysis of motor, mood, and cognition, which are commonly affected in neurodegeneration5. We have applied this battery to study the behavioral changes in young adult C57BL/6N mice after respiratory exposure to silica nanoparticles for 1 month and 2 months18. The results revealed that C57BL/6N mice exposed to silica nanoparticles showed various behavioral changes after different exposure times18. Briefly, results in the open-field test (Figure 2A) and the accelerating rotarod test (Figure 2B) demonstrated that silica nanoparticles exposure did not affect the locomotor or motor function in mice, indicating a full capability of accomplishing the other tests. Social interaction activity was affected after a 1 month exposure to silica nanoparticles (Figure 2C). Considering anxiety or depression would also decrease sociability, we analyzed data of the open-field test, elevated plus maze test (Figure 2D,E), and the forced swim test (Figure 2F), which did not indicate any comorbidity of anxiety nor depression at the 1 month time point. A 2 month exposure to silica nanoparticles resulted in anxiety according to the results in the elevated plus maze test (Figure 2E). A similar trend was shown in the central area duration in the open-field test (Figure 2D). Cognitive impairment was also detected in the Morris water maze test and novel object recognition test after a 2 month exposure (Figure 3). It should be noted that the protocol was slightly different in the two trials of the Morris water maze test. An additional lamp was added in the second trial, so all the mice always stayed under the lamp to keep warm. Hence, no nonperformer was shown in the second trial, whilst two out of eight mice became nonperformers in the probe test in the first trial.
We adjusted the protocol so that most of the tests in the battery can be repeatedly tested. The key is to maintain the motivation of the tests. Tests like social interaction test, novel object recognition test, and elevated plus maze test are motivated by novelty (i.e., a novel juvenile helper, novel objects, and a novel environment, respectively). By maintaining the novelty in the protocol, the young adult C57BL/6N mice showed a consistent performance when tested again after 1 month. According to our data, when introduced to two different helpers in the two trials, mice consistently showed a preference greater than ten-fold to the helper than to the empty chamber in the social interaction test (Figure 4A). In the novel object recognition test, the normal mice consistently preferred the novel object to the old object (Figure 4B). However, in the elevated plus maze test, when tested again in the same environment after 1 month, the exploration dropped by half (Figure 4C)10. Theoretically, young adult C57BL/6 mice can be tested repeatedly in these tests as long as the experimental condition, including the novelty and the status of the mice, remains the same. We have repeated these tests every month for up to three times in our lab. Noteworthy, the Morris water maze test cannot be tested repeatedly in the same group of young adult C57BL/6N mice as the experience significantly interferes with the performance when repeatedly tested. According to our data, the mice still remembered the platform even after 1 month, showing correct and long-term spatial memory. When changing the position of the platform, the experienced mice learned faster than naïve mice, as they had learned the rules and searching strategy from the prior training (Figure 4D).
Figure 1: Schematics. (A) Arrangement of the behavioral test battery and the schematic plots of (B) the social interaction test, (C) the elevated plus maze test, and (D) the novel object recognition test. Abbreviations: R = accelerating rotarod test; OF = open-field test; EPM = elevated plus maze test; NOR = novel object recognition test; SI = social interaction test; MWM = Morris water maze test; FST = forced swimming test. The starting point of the mouse in the test is shown by the mouse in the scheme. Please click here to view a larger version of this figure.
Figure 2: Changes in motor and mood in mice exposed to silica nanoparticles for 1 month or 2 months, detected by the behavioral test battery. Mice were tested in (A and D) the open field test, (B) the rotarod test, (C) the social interaction test, (E) the elevated plus maze test, and (F) the forced swimming test comprised in the battery. N = 8, 12, or 20, which means each group had 8, 12, or 20 mice, respectively, as demonstrated in each figure. N = 17 – 20 means each group had 20 mice, except for a control group at 1 month, which consisted of 17 mice. In panels A, D, and E, data were first normalized to control at each time point and, then, were analyzed with two-tailed Student's t-test. The data in panel B were analyzed by repeated measures two-way ANOVA. The data in panels C and F were analyzed with two-tailed Student's t-test. All data is shown as mean ±S.E.M. * and **** mean p < 0.05 and 0.0001, respectively. These data have been published previously by You et al.18. Please click here to view a larger version of this figure.
Figure 3: Changes in cognition after being exposed to silica nanoparticles for 1 month or 2 months. Changes in the mice's cognition after being exposed to silica nanoparticles for (A) 1 month or (E) 2 months, detected by the novel object recognition test. Changes in the mice's cognition after being exposed to silica nanoparticles for (B – D) 1 month or (F – H) 2 months, detected by the Morris water maze test. Mice were repeatedly tested in the novel object recognition test. A different batch of mice was tested in the Morris water maze test at different time points. N = 6, 8, 12, or 20, which means each group had 6, 8, 12, or 20 mice, respectively, as demonstrated in each figure. In panels A, C, D, E, G, and H, data were analyzed with two-tailed Student's t-test. The data in panels B and F were analyzed by repeated measures two-way ANOVA. All data are shown as mean ±S.E.M. * means p < 0.05. These data have been published previously by You et al.18. Please click here to view a larger version of this figure.
Figure 4: Representative data in tests. Representative data in tests, including (A) the social interaction test, (B) the novel object recognition test, and (C) the elevated plus maze test, tested in naïve mice (trial 1) and repeatedly tested in the same batch of mice (trial 2). (D) Representative data of the Morris water maze test when repeatedly tested. This figure has been modified from You et al.18. All data are shown as mean ±S.E.M. and analyzed with unpaired Student's t-test. P < 0.001, compared to trial 1. Please click here to view a larger version of this figure.
chambers in social interaction test | home made | (8 cm (L) x 6 cm (W) x 12 cm (H)), transparant with holes, plastic | |
cylindrical tanks used in forced swimming test | home made | 30 cm height, 20 cm diameters, glass | |
elevated plus maze | home made | open arms (30 x 5 x 0.5 cm) ,closed arms (30 x 5 x 16 cm), center platform (5 x 5 x 0.5 cm), 40 cm tall. Plastic, nontransparant | |
IITC Roto-Rod Apparatus | IITC life science Inc. | 755, series 8 | |
open field arena | home made | 60 cm (L) x 60 cm (W) x 40 cm (H), plastic, nontransparant | |
water maze | home made | 120 cm in diameter, 60 cm deep, steel |
Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.
Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.
Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.