Behavioral analysis in mice provided important contributions in helping understand and treat numerous neurobehavioral and neuropsychiatric disorders. The behavioral performance of animals and humans is widely different among individuals but the neurobehavioral mechanism of the innate difference is seldom investigated. Many neurologic conditions share comorbid symptoms that may have common pathophysiology and therapeutic strategy. The forced swim test (FST) has been commonly used to evaluate the “antidepressant” properties of drugs yet the individual difference analysis of this test was left scantly investigated along with the possible connection among other behavioral domains. This study conducted an FST-screening in outbred CD-1 male mice and segregated them into three groups: high performers (HP) or the active swimmers, middle performers (MP), and low performers (LP) or floaters. After which, a series of behavioral experiments were performed to measure their behavioral responses in the open field, elevated plus maze, Y maze, three-chamber social assay, novel object recognition, delay discounting task, and cliff avoidance reaction. The behavioral tests battery revealed that the three groups displayed seemingly correlated differences in locomotor activity and novel object recognition but not in other behaviors. This study suggests that the HP group in FST has higher locomotor activity and novelty-seeking tendencies compared to the other groups. These results may have important implications in creating behavior database in animal models that could be used for predicting interconnections of various behavioral domains, which eventually helps to understand the neurobiological mechanism controlling the behaviors in individual subjects.
Behavioral neuroscience, also known as biological psychology (Rosenzweig
There is no absolute and unified structure in assessing animal behaviors but some scientists categorized the behaviors into broad domains with corresponding behavioral test methods that can be performed (Karl
The forced swim test (FST) is one of the most common behavioral tests performed in rodents to evaluate the antidepressant potential of drug candidates. The mouse version of the forced swim test is a relatively short and low-cost behavioral test that requires no training of the mice and can be performed with minimal equipment. The mice that display high immobility throughout the trial period are interpreted as showing despair-like or depression-like behavior (Can
More generally, behavioral variability within a species is typically neglected in behavior research. The high variations in experimental results from individual subjects could be a big obstacle to various researchers but it may also remind the outstanding point that behavioral individuality needs to be accepted as part of research variables. Intriguingly, the identification of variability in the immobility profile of control mice in the FST could be one good starting point for the study of behavior domain interconnections. Considering the scarcity of study on the possible connection of FST responses to other behavioral domains, this paper targets to find out whether the divergent responders in FST could be classified and would also be accompanied by phenotype-dependent differences in other behavioral domains.
Male CD-1 mice (7 weeks old) were delivered from OrientBio (Seongnam, Korea). They were housed in a Plexiglas cage in groups of 10 and kept in a temperature- (22 ± 2°C) and humidity-controlled (55 ± 5%) animal room on an artificial 12–12 h light/dark cycle with lights on at 7 am. Habituation for a week in the new environment was done prior to initiation of experiments. Food and water were freely available except during the experiment and a special arrangement of behavioral tests. All experiments were performed between 09:00 to 16:00 and a minimum of 30 min acclimatization to the testing room was observed prior to every behavioral test. The whole behavioral study lasted for 6 weeks. All procedures and animal treatment were carried out in accordance with the Principles of Laboratory Animal Care (National Research Council, 2010) and were approved by the Animal Care and Use Committee of Konkuk University (KUIACUC), Korea (KU18054). All efforts were made to reduce the number of animals.
All mice were identified by tail markings before initiating the FST. Subject mice were allowed to swim in transparent cylindrical Plexiglas tanks (30 cm in height×20 cm in diameter) filled with 15 cm-deep water. After carefully putting each mouse in the tank, the timer and video recorder were simultaneously started. Each trial lasted 6 min and the last 4 min were used for analysis. At the end of each test, animals were removed from the water, dried with a paper towel and transferred back to their home cages. The immobility duration was scored for each mouse similar to the technique used by the previous report (Can
After the manual analysis of the video recording, the mice were classified into three groups: high performing (HP) or active swimmers characterized by an immobility profile lower than the standard deviation of the mean, middle performing (MP) with immobility duration nearest to the mean, and low performing (LP) group which has an immobility duration more than the upper standard deviation (Fig. 1). The identified groups were then housed together per group in a new cage and were allowed to habituate for a few days before starting the next behavioral tests. Most importantly, each group is evenly represented throughout the whole experiments performed.
Open field test was performed to evaluate both exploratory and locomotor activity and provide an initial screen for anxiety-related behavior in rodents. At the start of OFT, each study subject was gently placed in the center area of five large square chambers (42 cm×42 cm) and allowed to explore the arena for 25 min. An automated tracking system that used EthoVision software automatically analyzed the movements of the subject mice including the distance moved and movement duration as well as the time spent in the center area.
EPM assesses the anxiety responses of rodents in an elevated open platform (Pellow
Y maze is commonly used to assess the spatial working memory in rodents using the spontaneous alternation task (Wolf
The three-chamber social test is a method to evaluate the social ability of a subject mouse by exploring a stranger mouse in a wire cage over the empty cage, and preference to a novel stranger mouse over an already familiar one (Moy
The NORT test gauges the exploration of the subject mouse to a novel object over a familiar one without the presence of rewards to extract their natural propensity for novelty (Baxter, 2010). The test was conducted in an open field arena (42×42 cm) with two kinds of objects, generally consistent in height and volume, but different in shape and appearance. We followed the protocol from (Ennaceur, 2010) with modifications according to the laboratory condition. During the habituation period, the animals were allowed to explore an empty arena for 10 min. Then, two identical objects were diagonally placed at an equal distance from the nearest corner of the arena to familiarize the subject mouse to these objects for another 10 min. One familiar object was then replaced with a novel object and the subject mouse was again allowed to explore the objects for 5 min to test the short-term recognition memory (Tanaka and Curran, 2001). The time spent exploring each object was counted and the recognition between familiar and novel object was calculated as previously described (Vogel-Ciernia and Wood, 2014).
DDT is a measure of impulsivity in animals with the principle of forgoing small and immediate rewards to gain greater rewards at a later time (Matta
During the first 2 days of the training phase, the test subjects were introduced into the chamber with only the right aperture. Each nose poke on the aperture delivers a pellet. On days 3 and 4 of the training phase, the chamber is modified and will contain only the left aperture while on day 5, the subjects were introduced into a chamber containing both the left and the right apertures. It is best to start the test phase proper after 1 trial of having both the left and right apertures because the test subjects may develop a side preference the longer they are exposed to a chamber with two apertures. The left aperture would deliver one pellet instantly and the right aperture would deliver 5 pellets and illuminate the stimulus light. Over the course of the test phase, an increasing delay per day (0 s 10 s 30 s 50 s) is applied on the right lever’s delivery of the pellets. The computer program will tally the times the test subjects poked their nose on the corresponding apertures (Mitchell, 2014).
The cliff avoidance reaction is also used to assess the impulsivity of mice using a round plastic platform (diameter, 20 cm; thickness, 2 cm) elevated to a height of 50 cm (Yoshida
All collected data were expressed as the mean ± the standard error of the mean (SEM) and the GraphPad Prism software was used for statistical analysis. The normality in the baseline FST behavioral data was derived using the D’Agostino-Pearson test and a
The FST performed on 8-week old outbred CD-1 male mice produced a significantly wide range of response segregating them according to the immobility time during the 6-min trial period (Fig. 1). The high-performing group is considered active swimmers with 50–100 s immobility time during the test. The low-performing group, on the other hand, spent floating for about 200–230 s. The middle-performer having a mean of 150-s immobility were also obtained to serve as a midline reference group throughout the whole series of behavioral study.
To determine whether there are differences in locomotor activity among FST-screened mice, an open field test was performed (Fig. 2). There were significant differences among groups in one-way ANOVA of the two parameters in the OFT (Fig. 2A, 2B). Tukey’s post hoc comparison revealed that the HP group showed higher distance moved (Fig. 2A) and movement duration (Fig. 2B) compared to LP but not with MP group. The time spent in the center area is not different between the groups (Fig. 2C). The immobility time in FST and distance moved in OFT showed a negative correlation pattern among three groups suggesting these two behaviors might have common regulatory components (Fig. 2D).
To determine a possible relationship between the immobility profile in the FST and anxiety-like behavior, we performed the EPM test. However, the results showed no significant difference among the three groups both in the percentage of time spent in the open arms (Fig. 3A) and the frequency of entry in the open arms (Fig. 3B).
To investigate whether the variable responses in FST is linked to spatial working memory through the measure of arm entry alternations, Y maze test was performed (Fig. 4). All groups showed a similar number of arm entries (Fig. 4A) during the trial and no significant difference was also found in the one-way ANOVA between groups when the spontaneous alternations (Fig. 4B) were calculated.
To find out whether differences in FST performance can lead to differences in social behavior profiles, we performed three-chamber social assays (Fig. 5). In the sociability test, no difference was observed in all parameters that were analyzed including the duration in each compartment (Fig. 5A), approach duration (Fig. 5B) and sociability index (Fig. 5C), although the sociability index of HP group tends to be higher than MP and LP groups. In the same way, social preference for novelty was also identical in all groups from all parameters of the duration in compartment (Fig. 5D), approach duration (Fig. 5E) as well as the social preference index (Fig. 5F).
Looking at object exploration profile, we observed that the HP group in the FST displayed an increased exploration time to the novel object which was significantly different compared to MP (
The percentage of choice for the large reward in the delay discounting task was similar between the three groups (Fig. 7). In the cliff avoidance reaction, most of the mice didn’t jump out of the edge during the 10-min trial showing no inclinations of impulsivity in these animals. In measuring the time spent and head dipping around the edge area of the platform, there were tendencies of higher values in the HP compared to both MP and LP groups but not significantly different from each other (
The state of the mind, emotion and physical actions are interconnected and are often complex if not simple. A number of parameters used in behavioral tests such as FST assess the animal response in terms of their actions, which could be affected by various parameters such as neurophysiological and emotional factors of individual subjects. At present, the vast majority of literature interprets the results of FST as being associated with depression or the so-called “depression-like behavior”. For this reason, Commons challenged the idea of assuming a connection between animal behavior and human psychopathology that discourages critical thoughts. Rather, the FST is an interesting and unique test that contributes to the understanding of neural networks regulating the behavioral response towards the acute stressful situation, which may be defective in many neurological disorders such as depression, ASD, and other disorders (Commons
A fundamental inter-strain difference of response in the FST has already been established (Petit-Demouliere
The results in the OFT revealed that the HP mice in FST (that is the active swimmers) also traveled more distance in the open field arena compared to LP mice. This result confirms previous findings in a reciprocal fashion where the grouping was done with the response in OFT before doing the FST (Jama
Another interesting finding is the increased novelty-seeking behavior of HP mice as evidenced by more time spent in the novel object and higher recognition index especially compared to the LP group. This result introduces the connection of active swimming in the FST with novelty-seeking behaviors and that these behaviors can affect each other in various situations and treatment conditions. Our previous study suggested that high novelty-seeking responses in NORT as well as in OFT in rats would also respond more positively to certain drug treatment (dela Peña
Although it is unclear what is the underlying mechanism governing the individual difference in behavioral performance in this study as well as the neural substrates determining the interconnected regulation in some domains of the behavioral paradigms, it is obvious that we can take advantage of the observed individual inter-correlation of the behavior to excavate genes and proteins involved in the regulation of the behavior. Future study will also tell us whether the inter-correlative behavior will be maintained through the neurological, pharmacological, physical and psychological perturbation to the individual subjects, a theme closely related to the personalized brain functional landscape.
The authors do not have any conflict of interest regarding this study.
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (NRF-2017M3A9G2077568).