Chapter 5

The Forced Swim Test for Depression-Like Behavior

in Rodents
Ann-Katrin Kraeuter, Paul C. Guest, and Zoltán Sarnyai

Abstract
The forced swim test assesses learned helplessness, which is a feature of depression-like behavior in rodents.
This test has also been used in testing the efficacy of existing and novel antidepressant drugs. It is based on
the natural tendency of rodents to escape from water. Rodents are placed in a cylinder filled with water and
the presumption is that those with a depression-like phenotype give up swimming earlier than those that are
not depressed. Furthermore, antidepressant drugs reverse this effect. This chapter describes the basic setup
and conduction of the test, along with interpretation of the results. It should be emphasized that this test
should be conducted as part of a series of behavioral assessments in order to increase the accuracy of the
results.

Key words Animal model, Depression, Forced swim test, Behavior, Biomarker, Drug screening

1 Introduction

Validated animal models and tests of depression-like behavior are

crucial for understanding the underlying pathophysiology of this
psychiatric disorder and for assessing the efficacy of existing and
novel antidepressant drugs. One test which has received consider-
able attention is the forced swim test, which was originally designed
by Porsolt and co-workers [1]. The forced swim test is based on the
finding that rodents immersed in a cylinder filled with water
attempt escape but show increased periods of inertia after initial
endeavors, when they learn that escape is impossible (Fig. 1). Then,
the duration of time for the transition to immobility or the total
time spent immobile is used an index of the learned helplessness or
depression-like behavior (Fig. 2).
The ease of setting up of the forced swim test leads to high
reproducibility both within and across different laboratories,
making it particularly suitable as a pre-clinical model and for drug
screening attempts [2–4]. One problem is that the results of this
test alone do not necessarily give an indication of low mood or

Paul C. Guest (ed.), Pre-Clinical Models: Techniques and Protocols, Methods in Molecular Biology, vol. 1916,
https://doi.org/10.1007/978-1-4939-8994-2_5, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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76 Ann-Katrin Kraeuter et al.

Fig. 1 General setup and behaviors of mice during the forced swim test

Fig. 2 Graphs showing the effects of antidepressant drugs on latency to first inertia and time spent inert in the
forced swim test. Cont control (vehicle) and Fluox fluoxetine

depression, but, more simply, it gives a readout of the time taken to

give up a fruitless struggle [5]. It is now accepted that explicit
guidelines are required and multiple tests should be carried out in
series in order to more fully characterize the behavioral phenotype
of an animal [6, 7]. We and other researchers are advocating that
multiple tests are required as this approach can give a more com-
plete profile of animal behavior [7].
Here we report a protocol for conduction of the forced swim
test as a component of such a behavioral test series. The main focus
is on the preliminary steps of animal pre-adaptation that should be
 Forced Swim Test 77

taken to improve the reproducibility and accuracy of the test. In

addition, we present the materials required and the actual test
procedure, along with post-test interpretation of the results. A
number of factors should be considered that can affect the test
results such as age, weight, and gender of the animals as well as
environmental conditions such pre-handling, social environment,
and dietary conditionsfor each animal tested (for review see [8]).
The experimental observer and the physical environment can also
affect the results and this factor will also be considered.

2 Materials

1. Test and control mice (see Note 1).

2. 2 L beaker.
3. Warm water (see Note 2).
4. Webcam/camera mounted on a retort stand.
5. Test room with adjacent operator room (see Note 3).
6. Scales for weighing the animals.
7. Timer.

3 Methods

3.1 Experimental 1. Acclimatize mice at least 7 days prior to the experimental

facility and the operator (see Note 4).
2. On the morning of behavioral study, record the body weight of
the test animal in conjunction with relevant metadata of the
study.
3. In the test room, place the camera into the clamp of the retort
stand.
4. Ensure that all beakers are clean prior to testing by rinsing with
tap water and wiping with a paper towel.
5. Fill the beaker with 1.4 L warm tap water and record the
temperature (see Note 2).
6. Connect the camera to acomputer in the adjacent room via
along USB cable.
7. The operator should ensure that all areas of beaker can be seen
on the computer screen.
8. One hour before testing, place the animal in the behavioral
testing room (see Note 5).
9. After 1 h, start filming (see Note 6).
10. Set the timer for 6 min.
78 Ann-Katrin Kraeuter et al.

11. Place the test animal into the beaker, holding it gently by the
base of the tail.
12. Leave the test room.
13. Press start on the timer.
14. Allow the animal undisturbed time inside the beaker (see Note
7).
15. Stop the recording after 6 min.
16. Remove the animal from the beaker, gently wipe off excess
water with a tissue, and place back into the home cage.
17. Put fresh tissues in the cage, and make sure that no breeze from
the air conditioning gets into the cage (see Note 8).
18. Return to the computer and save the video recording.
19. Empty and rinse the beaker before proceeding to the next
animal.
20. Refill with clean warm water and check the temperature as
described above.
21. When the experiment is completed, clean all equipment and
pack away.
22. Place all animals back into the holding room taking the same
steps as above.

3.2 Data Analysis 1. By re-examining the video, hand score the behavior of the
mouse over the 6 min interval taking into account: (1) time
to first immobility; (2) time spent mobile; and (3) time spent
immobile; (4) bouts into mobility; (5) bouts into immobility
(see Note 9).
2. The scoring should also be performed by another experimenter
who is blind to the experimental conditions (see Note 10).

4 Notes

1. This could be an experimental manipulation [9, 10] or a

genetic cause [11] that either induces or is associated with a
depressed, anxious, or fearful phenotype. The experimenter
should ensure that all procedures for work with animals have
been approved by the relevant institutional bodies.
2. The temperature of the water should be comfortable for the
test animals so that this is not a factor in the test outcome. We
favor the use of water that is at room temperature (22–26  C).
3. Separate rooms are needed for conduction of the actual test and
for the operator to ensure that the operator has no influence on
the test outcome.
 Forced Swim Test 79

4. This prior exposure and handling of the animals has been found
to lead to more reproducible results as it helps to minimize the
effects of potential confounding factors [7, 12]. No culling of
animals or blood collection should take place in the facility at
least 24 h prior to behavioral testing. Low ambient noise
should be maintained throughout behavioral testing.
5. Again, this helps the animals to acclimatize and reduce stress
for more reproducible results.
6. The operator should record all details of the experiment includ-
ing the animal identification code, the experimental manipula-
tion, drug type (if applicable), name of the experiment, etc.
7. During this time, the animal should be monitored closely by
the operator in the adjacent room on the computer screen. The
experimenter should not leave the screen unattended for any
reason. If the mouse sinks under the surface of the water, the
test should be stopped immediately and the animal should be
removed and tended to ensure its safety. The mouse should also
be wiped with a tissue to remove any excess water and placed
back into its home cage to recover. At this stage, the experi-
menter should ensure that there is no breeze on the animal and
that it has recovered sufficiently from the experience.
8. The welfare of the animal should always come first.
9. The general interpretation is that a decreased time to first
immobility, decreased mobility time, or increased immobility
time, shows a learned helplessness or depression-like behavior.
However, it should be noted that this may not be distinguish-
able from other possible behaviors as stated above and by other
researchers in the field [5, 6]. Thus other tests should be
carried out in the form of a battery to more accurately assess
the behavior of the animal [7].
10. The inter-rating accuracy should be >80%.

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