1Department of Psychiatry, University of Maryland School of Medicine, 2Tulane University School of Medicine, 3Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 4The Program in Neuroscience, University of Maryland
This article is a part ofJoVE Neuroscience. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.Recommend JoVE to Your Librarian
Current Access Through Your IP Address
Current Access Through Your Registered Email Address
Can, A., Dao, D. T., Arad, M., Terrillion, C. E., Piantadosi, S. C., Gould, T. D. The Mouse Forced Swim Test. J. Vis. Exp. (59), e3638, doi:10.3791/3638 (2012).
The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states. Mice are placed in an inescapable transparent tank that is filled with water and their escape related mobility behavior is measured. The forced swim test is straightforward to conduct reliably and it requires minimal specialized equipment. Successful implementation of the forced swim test requires adherence to certain procedural details and minimization of unwarranted stress to the mice. In the protocol description and the accompanying video, we explain how to conduct the mouse version of this test with emphasis on potential pitfalls that may be detrimental to interpretation of results and how to avoid them. Additionally, we explain how the behaviors manifested in the test are assessed.
1. Materials and Method
1.1. The water tanks
The cylindrical tanks (30 cm height x 20 cm diameters) required for the mouse forced swim test (FST) in our laboratory are constructed of transparent Plexiglas, as this material is able to withstand the frequent movement of the tanks and accidents better than glass. The water level is 15 cm from the bottom and should be marked on the tank to ensure that the volume of water is consistent across mice. The number of tanks should ideally be at least twice as many as the number of mice being tested at a time, so that the second water tank set can be filled while the first set is in use. The dimensions of the tanks should be selected in a way that the mice will not be able to touch the bottom of the tank, either with their feet or their tails, during the swimming test. The height of the tank should be high enough to prevent the mice from escaping from the tank. Please note that the diameter of tank and the depth of water are important parameters that can be adjusted to change the behavior of mice (for a detailed analysis of these issues see1-3).
A water resistant infrared thermometer is preferable, since rapid measurement of temperature reduces the amount of time required to conduct the test. However, a glass mercury thermometer will also be sufficient for this task.
1.4. Video recording device
We use a video camera supported by a tripod. Since this test usually involves multiple animals being tested at the same time, live scoring will be very difficult and is not advisable. The video camera should record in high enough resolution to render a quality picture that will be used later for behavioral scoring. Always make sure there is sufficient recording memory in the camera before starting the test. We use a video camera that records digitally without the use of mechanical media (i.e. video cassette), allowing for digital transfer of videos. If there are excessive reflections on the tanks, which may occur in laboratory environments with overhead fluorescent illumination, you may want to use a polarizing lens filter with your camera.
In our lab we have two sets of dividers (35 cm height x 22 cm width x 22 cm depth). These are rectangular with three walls and are used as both background and as dividers between tanks to prevent mice from seeing each other during the test and potentially altering their behaviors. One set can be black for albino and light colored animals; the other set can be light colored for dark colored animals in order to render high contrast. The experimenter should make sure that the surfaces of the dividers are not overly reflective so that they alter camera images, or render major differences between illumination levels.
1.6. White noise generator
This is needed in laboratory environments in which sudden loud noises can be heard that would potentially startle the animals. The noise generator will mask such intermittent disturbing sounds. The volume level of the white noise generator should be selected to be above other ambient and unexpected noises. In our experimental room the ambient noise level (without the white noise generator activated) is 60 dB. The total noise level with the white noise generator activated at the location where the tanks are placed is 70-72 dB. However it should be noted that these figures are provided as example only, and each laboratory should select the right noise levels according to their unique environment and circumstances.
1.7. Drying paper and heat lamp
Before returning the animals to their home cages, it is important to dry them gently using paper towels and it is helpful to use a heat lamp (be certain the exposure temperature does not exceed 32°C) to prevent hypothermia.
2. Behavioral Procedures
3. Behavior Analysis
4. Representative Results
There are marked differences between genetically distinct inbred and outbred mouse strains in terms of their baseline immobility and responding to a specific drug5-11. For example, we identified differential antidepressant-like responses to lithium in a panel of mouse strains (Figure 1)5. Experimental details of this experiment are published in Can et al., 20115.
Figure 1. Immobility time (in seconds) in the forced swim test, five hours after a single i.p. injection of saline, 200, 300, or 400 mg/kg in various inbred and outbred mouse strains. *:p<0.05, **:p<0.01, ***:p<0.001 denote a significant difference compared to saline group, Dunnett's post hoc test. Data are expressed as mean ± SEM. Number of animals per group for each strain is 6-8 (Figure reproduced from5).
Not all mouse strains are suitable for the FST. Some strains, such as Black Swiss, NIH Swiss, and FVB/NJ show little or almost no immobility under control conditions, therefore representing a floor effect (Figure 1)5. The lack of baseline immobility effectively prevents detecting an anti-depressant effect of experimental manipulations. It is also possible, while very rare, that some mouse strains may behave aberrantly and dive into the tank during the test even though they can float. One such strain is DBA/1OlaHsd (unpublished observation in our laboratory). Such strains are not suitable for the FST. Because of this diving risk, however small, when testing a new strain that has not been previously tested in the FST or a mouse harboring a novel genetic manipulation, it is imperative to carefully observe the initial trials to rescue mice if they engage in potentially harmful behaviors.
In the experimental design described here, multiple animals (up to five) are tested at the same time. While the dividers we use prevent mice from seeing each other during the test, and the white noise generator suppresses audible vocalizations, our set-up does not prevent all ultrasonic or olfactory cues from being transmitted. Though unlikely given the nature of the test, these could affect the behaviors of mice. One solution to this problem would be to test the animals individually. However, this approach has its own problems. For example, commonly, the animals tested in each session come from the same homecage. This allows randomization and counterbalancing of the experimental variables. Testing mice individually would mean removing one mouse at a time from the homecage. This will cause repeated stress and disturbance of social hierarchy in the cage among the others left behind. Another issue with testing singly are the time constraints. Testing one mouse at a time will extend the experiment into many hours resulting in a situation in which mice are tested at different times of the circadian cycle. This may create confounding time of day effects. The researchers should keep these issues in mind while designing their experiments.
The FST (sometimes called Porsolt swim test) was developed first for rats and then modified for mice by Porsolt and colleagues12,13. In addition to the above-described protocol successful in our laboratory, a number of largely subtle test modifications have been published (see Hascoët and Bourin for a complete review1). It is a common test used for evaluation of the efficacy of anti-depressant drugs and the effects of various behavioral and neurobiological manipulations in basic and preclinical research3,14-16. It has been described as rendering a situation in which "behavioral despair" is induced; that is, the animal loses hope to escape the stressful environment13. 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 conducted with minimal equipment. This is in contrast to the rat version of the test, which generally involves exposure to the water tank one day prior to the test day17 .
Because of its popularity there is a wealth of data regarding the effects of various antidepressants in the FST. This allows researchers to compare and contrast their own results with others (see Hascoët and Bourin for 2009 review1). These characteristics of the FST make it an important tool in academic research and drug discovery in industrial settings where reliability and high throughput screening of novel compounds are essential. An additional feature of the FST is the availability of commercial automated behavior analysis systems that can accelerate the data collection process18-20. However, in our experience, these automated systems require extensive validation by human scoring. Additionally, automated parameters may have to be readjusted when using different strains, especially when the level of background contrast changes, or with mice of different sizes or behavioral responses.
Another area in which the FST is used is neurogenetic research in which the genetic basis of depression-related behaviors is investigated. These types of studies involve comparison of various mouse strains with or without the use of anti-depressant drugs and comparisons of genetically modified or selectively bred mice and their wild type counterparts6,21-23. In this regard, the FST has proven to be useful in basic research related to the neurobiology and genetics of mood disorders. However, the FST is not a full spectrum analog of human depression. Even though there are exceptions, the FST has a considerable level of predictive validity, since it is reasonably sensitive to compounds that are effective in humans as anti-depressants and insensitive to those that are not effective24,25. Since the behavioral outcome of the FST is one-dimensional it can only indicate the antidepressant efficacy of compound or experimental manipulations, but it cannot differentiate mechanistic differences between them. This is in contrast with the rat version of the FST, where rats manifest both swimming and climbing behaviors that can differentiate between serotonin and norepinephrine acting compounds26. Also any manipulations that may affect the overall activity levels may potentially alter immobility in the FST leading to spurious conclusions. Therefore it is important to verify the results of FST with separate behavioral tests that measure overall activity such as the open-field test1,27. It is beneficial to keep in mind that the FST does not represent the human condition, and to extent which underlying neurobiological mechanisms of the behaviors manifested by model animals in the FST and human depression overlap is not entirely clear28. However, these types of limitations should not devalue the usefulness of FST as a drug discovery and validation tool.
Authors declare no conflicts of interest.
This study has been supported by the grant NIHM R01 MH091816 and R21 MH084043 to TDG.
|White Noise Generator (optional)|