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The forced swim test (FST) is one of the most commonly used animal models for assessing depressive behaviors. The forced swim test involves the scoring of active movements such as swimming and climbing vs. passive immobile behavior while swimming in a cylinder from which there is no escape. A wide range of treatments has been shown consistently to reduce the amount of immobility time while increasing active escape behaviors.
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The Forced Swim Test (FST), created by Roger D. Porsolt in the late 1970s, serves as a straightforward method to evaluate the antidepressant potential of substances in small animals. Depression, a widespread mood disorder affecting millions worldwide, remains not entirely understood in its origins. Mimicking depressive symptoms in animals proves challenging, but certain behavioral cues offer a basis for assessing the efficacy of drug therapies and other interventions.
Also referred to as the Behavioral Despair test, the Forced Swim Test induces learned helplessness by placing the animal in a stressful situation—a water-filled cylinder with no means of escape. The animal, realizing its predicament, adopts a posture of immobility, expending minimal effort to keep its head above water. Treatment typically results in increased efforts to escape. Early cessation of swimming behavior is akin to human experiences of hopelessness amid depressive symptoms. The test’s behavioral parameter aims to evaluate antidepressant effects based on the premise that stressful life events contribute significantly to depression and related behaviors.
The apparatus comprises a transparent Plexiglas cylinder filled with water at a level that prevents the subject from touching the floor or climbing over the walls for escape.
Since its inception and adoption by Porsolt in the late 1970s, the Forced Swim Test has undergone minimal alterations in both its apparatus and protocols.
In a 1987 study, Shimazoe et al. proposed a modification involving the placement of detectors on the cylinder walls. These detectors could capture and record vibrations generated by the subject’s movements during the test, offering an objective measure of immobility associated with behavioral despair.
Another enhancement was proposed by Lucki in 1997. They recommended increasing the water depth in the apparatus and utilizing time-sampling techniques to assess animal behaviors. This approach enabled the classification of behaviors into climbing and swimming categories, facilitating the quantification of escape behavior. Additionally, researchers could discern differences in behaviors induced by serotonin reuptake inhibitors.
2.1 Origin
Roger D. Porsolt introduced this test in the late 1970s through several publications (Porsolt et al.,1977a, Porsolt et al.,1977b, Porsolt et al.,1978). He and his team demonstrated that rodents could mimic depressive states and respond to antidepressant drugs. When they gave these drugs to rodents before the forced swim test, they noticed a reduction in despair and immobility (Porsolt et al.,1977a). Later on, Porsolt compared different mouse strains’ behavior in this test, with and without antidepressant drugs (Porsolt et al.,1978). Since these early studies, the forced swim test has become a widely accepted method for testing antidepressant compounds.
2.2 Developments
In their 1981 paper, Herman et al. examined how different compounds affected rat behavior in the Forced Swim Test. They also explored the relationship between test performance and the rats’ natural levels of exploration and movement. Their findings revealed that some compounds increased the time rats spent immobile, while others decreased it.
Shimazoe et al. proposed a way to objectively measure immobility in the Forced Swim Test by recording the vibrations caused by the rats’ attempts to escape and comparing it with their movements in an activity cage. They modified the test apparatus by adding sensors to the cylinder walls to detect vibrations when rats were exposed to different drugs.
Trzctńska et al. investigated the impact of prenatal protein malnutrition on female and male rats in the Forced Swim Test. They conducted two experiments: one where malnourished rats underwent the test before and after a stress regimen, and another with two groups of male rats, one subjected to stress and the other not.
2.3 Recent Developments
Researchers explored the potential antidepressant-like effects of a prominent amino acid found in fenugreek seeds on olfactory bulbectomized rats (Kalshetti et al., 2015). Results from the Forced Swim Test indicated that rats treated with the amino acid exhibited reduced immobility and increased swimming time.
Bergman et al. investigated whether deep brain stimulation could induce antidepressant-like effects in mice. Following deep brain stimulation of the ventromedial prefrontal cortex, both wild-type and knockout mice showed decreased immobility in the Forced Swim Test. This suggests that deep brain stimulation may offer a promising avenue for treating individuals with a dysfunctional serotonin transporter.
The Forced Swim Test involves a cylindrical tank ranging from 25 to 50 cm in height and 10 to 30 cm in diameter, designed to accommodate rodents and small primates. The tank is filled with water at room temperature, ensuring that the subject cannot touch the bottom with its paws or tail, thus preventing escape. Opaque dividers are commonly inserted into the tank to facilitate testing with multiple subjects.
While it’s possible to score behaviors live, it’s advisable to utilize video recording and tracking software like Noldus Ethovision XT or ANY-Maze to enhance accuracy and reduce scoring discrepancies. Adequate lighting is essential for optimal recording quality, and a white noise generator can help mask any extraneous sounds that might affect the subject’s performance.
The Forced Swim Test evaluates the depressive state of animals exposed to antidepressant treatment versus a sham control group by observing their responses in a stressful setting. Initially, subjects display efforts to escape the environment, but eventually, they enter a state of behavioral despair characterized by immobility with minimal movements to keep their heads above water. This sense of helplessness and immobility tends to decrease with antidepressant treatment.
The test requires minimal prior training of animals and should ideally avoid external stressors. The natural sequence involves an initial struggle followed by behavioral despair once animals are placed in the apparatus.
Typically, a six-minute testing period is employed, although durations may vary between 4 to 20 minutes depending on the species. The entire test is recorded, with analysis often focusing on the last four minutes. The initial two minutes are marked by high activity, potentially masking treatment effects. Thus, the emergence of despair in control animals marks the critical period for observing treatment effects.
Evaluation of Depressive-like States in Small Mammals
The Forced Swim Test procedure begins by filling the cylinder with clean, room temperature water to an appropriate level. Dividers, if used, are inserted at this stage. Test substances are administered to the subjects either 30 minutes before the test for injections (intraperitoneal or subcutaneous) or 60 minutes prior for oral administration. Video recording starts before placing the subject into the cylinder.
Subjects are gently lowered into the tank by their tails. Immobility behavior is scored during the final 4 minutes of the 6-minute session, which includes brief periods of minimal activity as the animal stays afloat.
After the session concludes, recording stops, and subjects are carefully removed from the tank by their tails, dried with towels, and placed under a heat lamp for warmth before returning them to their home cage.
If a subject fails to maintain swimming or floating behavior, it is promptly removed from the tank and excluded from the experiment.
Data from the forced swim test is typically visualized by depicting the duration animals spend engaged in various behaviors. This includes swimming, climbing, mobility, or immobility, which can be determined by analyzing experiment recordings either manually with a stopwatch or using a time-sampling method. In this method, the experiment is divided into five-second intervals, and the animal’s activity during each interval is noted.
In this test, mobility is broadly defined as any movement beyond what is necessary to keep the head above water (Cryan & Mombereau, 2004). Graphing the time spent mobile versus immobile allows for straightforward comparison between intervention and sham control groups.
Graphical comparisons illustrate the impact of antidepressant treatments: animals receiving saline typically exhibit increased immobility and reduced mobility, while those treated with antidepressants show decreased immobility and increased swimming and climbing. Control animals stimulated with motor activity, such as caffeine, tend to display heightened activity throughout the test.
Normally, groups of 10-15 animals are sufficient for obtaining statistically significant results (p < 0.05) using statistical tests like 1- or 2-way ANOVA, with post hoc analyses such as Dunnett’s or Bonferroni’s tests (Can et al., 2012; Costa et al., 2013).
Burgdorf et al.’s research highlighted the potential of rough-and-tumble play in developing new therapies for stress-related disorders. Their study revealed that subjects exposed to three minutes of rough-and-tumble play over three days displayed increased resilience to stress, as evidenced by their performance in the Forced Swim Test. Notably, these subjects showed a reversal of stress-induced effects caused by chronic unpredictable stress.
In a separate study, Yuen et al. sought to bridge the gap between data obtained from the Forced Swim Test and its relevance to human therapeutic doses. They conducted dose-response and time-course experiments using four commonly prescribed antidepressants, comparing the results to human dosage equivalents. Their findings suggested that adopting a concentration-based approach could enhance the prediction of clinically effective doses for potential new antidepressant agents.
The Forced Swim Test has proven highly effective in assessing the efficacy of antidepressant compounds, as evidenced by numerous published studies. Its extensive data collection, combined with its ease, reliability, and speed of execution, makes it an invaluable tool for drug discovery and comparative research across various subjects and compounds. Moreover, the test’s simplicity allows for simultaneous testing of multiple subjects using dividers, without requiring pre-training.
However, it’s crucial to recognize that the test itself induces significant stress on the subjects, which may influence their behaviors over repeated trials. While the test mirrors a specific type of stress-induced behavior associated with human depression, it’s essential to acknowledge that these behaviors stem from extreme stress.
Although the Forced Swim Test can induce a state of hopelessness akin to human depressive feelings, it’s important to remember its limitations. It doesn’t encompass the entire spectrum of human depressive states, and the extent to which the observed behaviors in animal models reflect human depression remains uncertain. The test provides a one-dimensional outcome and doesn’t elucidate the mechanisms underlying a compound’s antidepressant properties, except in rats where distinctions between various compounds can be discerned.
Moreover, the effects of tested compounds on overall activity levels may influence the reliability of Forced Swim Test results. Therefore, it’s crucial to monitor and control overall activity levels using supplementary tests such as the Open-Field test.
| Dimensions | N/A |
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| Species | Large Rat (above 500g), Mouse, Rat |
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