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NATURE PROTOCOLS | VOL.7 NO.6 | 2012 | 1009
INTRODUCTION
Major depression is a key health concern. In the United States, the
lifetime prevalence is estimated to be as high as 16.2% (ref. 1).
The World Health Organization states that it is the second-largest
global health burden2,3. Although there are pharmacological agents
available to treat depression, approximately 30–40% of patients
are unresponsive to them4, and those who benefit only do so after
a delayed onset of action. Therefore, a major emphasis in modern
psychiatric research is to uncover the underlying etiology of mood
disorders. It is hoped that this will help in the development of novel
efficacious antidepressant treatments.
A key component of such research is the use of animal models,
which are predictive of antidepressant activity. The FST, as origi-
nally reported by Porsolt and colleagues5–7, has developed into the
most widely used model for assessing antidepressant-like activity
in rats. This is largely due to its high throughput, ease of use, inter-
laboratory reliability and specificity. Moreover, it has been applied
to other species, including mice6,8–10, Mongolian gerbils11 and sand
rats (a species of gerbil)12.
Advantages and disadvantages of the FST
The FST is based on the observation that when rats are exposed
to water, after initial intense escape-directed behavior, such as
swimming and climbing, they stop struggling and show passive
immobile behavior. The immobile behavior is believed to reflect
either a failure to persist in escape-directed behavior after stress
(i.e., behavioral despair) or the development of passive behavior
that disengages the animal from active forms of stress coping13–16.
A wide range of antidepressant treatments have been shown
consistently to reduce the amount of immobility time in the test
by increasing active escape behaviors9,15,17. Although the FST is
sensitive to acute antidepressant treatment, a wide range of other
nonantidepressant compounds examined in the test are ineffec-
tive in altering immobility time14,17,18. However, stimulants, seda-
tives or motor-impairing compounds can lead to false-negative or
false-positive results in the test. Therefore, it is necessary to sepa-
rately assess locomotor activity in order to determine whether the
drug of interest alters general activity. For example, a stimulant,
while potentially decreasing immobility time in the FST, may also
increase locomotor activity in the home cage or locomotor activity
chamber, thus confounding the FST results. Therefore, in order
to ascribe a specific antidepressant-like effect to a treatment that
increases locomotor activity, further depression tests have to be
performed that do not rely on locomotor output in the final read-
out19. Similarly, a treatment that has sedative properties and a pro-
depressant-like effect in the FST would require additional testing
to separate the two parameters. This major limitation also applies
to other tests used in current basic psychiatric research, in that the
majority of established tests are influenced by drugs that modulate
the general activity of the animal being tested20. In general, many of
the currently available antidepressant compounds, while reducing
immobility time in the FST (i.e., an increase in active behavior),
concomitantly reduce general locomotor activity. This separation
highlights the different substrates underlying the FST and general
locomotion.
A further major criticism of the FST is that acute antidepressant
treatment is efficacious, whereas in the clinic chronic administration
is required, together with a delayed onset of action. However, this criti-
cism can also be leveled at the vast majority of current antidepressant-
sensitive preclinical models. Moreover, there is evidence that antide-
pressants can have rapid effects on a subset of symptoms21, as opposed
to, for example, the 2–4 weeks required to achieve a positive effect on
the Hamilton Depression Rating Scale (HAM-D) score. Furthermore,
doses of antidepressants that are ineffective acutely have been shown
to provide antidepressant-like effects after chronic administration22,23,
and chronic administration tends to be required to reverse chronic
stress-induced increases in immobility17 (and see below).
These criticisms (reliance on motor function and acute drug
administration) are common in tests of antidepressant efficacy, such
as the FST and the tail suspension test. However, as stated above,
these tests have high throughput and interlaboratory reliability. In
contrast, animal models that require an inducing manipulation,
such as olfactory bulbectomy, chronic stress paradigms and learned
helplessness, can be considered animal models of depression rather
than tests of antidepressant efficacy. Such models are responsive to
Using the rat forced swim test to assess
antidepressant-like activity in rodents
David A Slattery1 & John F Cryan2,3
1Department of Behavioural and Molecular Neurobiology, Institute of Zoology, University of Regensburg, Regensburg, Germany. 2Department of Anatomy and
Neuroscience, University College Cork, Cork, Ireland. 3Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland. Correspondence should be addressed to
D.A.S. (david.slattery@biologie.uni-r.de).
Published online 3 May 2012; doi:10.1038/nprot.2012.044
The forced swim test (FST) is one of the most commonly used animal models for assessing antidepressant-like behavior.
This protocol details using the FST in rats, which takes place over 48 h and is followed by the video analysis of the behavior. The
swim test involves the scoring of active (swimming and climbing) or passive (immobility) behavior when rodents are forced to
swim in a cylinder from which there is no escape. There are two versions that are used, namely the traditional and modified FSTs,
which differ in their experimental setup. For both versions, a pretest of 15 min (although a number of laboratories have used a
10-min pretest with success) is included, as this accentuates the different behaviors in the 5-min swim test following drug
treatment. Reduction in passive behavior is interpreted as an antidepressant-like effect of the manipulation, provided it does not
increase general locomotor activity, which could provide a false positive result in the FST.
© 2012 Nature America, Inc. All rights reserved.
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1010 | VOL.7 NO.6 | 2012 | NATURE PROTOCOLS
more chronic antidepressant treatment and may even use the FST
as a readout, but they have the disadvantages of lower throughput
and interlaboratory reliability (for a more detailed discussion of
advantages and disadvantages of animal models used in depression
research, see refs. 20,24).
Moreover, it is becoming apparent that in order to develop appro-
priate models of depression an endophenotypic-based approach
is required24–26. Depression consists of numerous symptoms, some
of which can be studied in rodents, such as anhedonia and sleep
alterations, whereas others clearly cannot, such as suicidal ideation.
Despite the presence of psychomotor agitation or retardation as a
symptom of depression, the FST does not fulfill an endopheno-
type approach given the necessity to control for general locomotor
activity. Altered stress coping is one of the hallmarks of depres-
sion27; thus, the test may be appropriate in order to shed light on
the underlying neurobiological basis of discrete aspects of the etiol-
ogy of depression despite lacking the validity to dissect other core
endophenotypes of the depression syndrome.
The FST (both the original and modified) in rats is also sensitive
to the pro-depressant effects of a number of stressors, including
prenatal stress28, chronic subordination stress29,30, chronic cor-
ticosterone administration31 (but see ref. 32) and amphetamine
withdrawal33. The fact that the FST gives bi-directional results to
common manipulations gives the model high predictive validity.
Thus, despite the lack of face validity, such evidence shows that the
FST is an appropriate tool for preclinical mood disorder research.
Experimental design
In rats, a preswim exposure, 24 h before the test session, is required
to discern antidepressant-like activity. The use of a preswim ensures
that the rats quickly adopt an immobile posture on the test day,
which enables the effect of the tested compounds to be more eas-
ily observed13,14,18,34. However, for unknown reasons, only a test
exposure (i.e., no preswim required) is sufficient in mice to ensure
a stable immobility baseline, which can be reduced with a broad
range of acute antidepressant treatment.
The influence of the strain of rodent on behavior in the swim test
has been more extensively characterized in mice than rats, where
more than a tenfold difference in baseline immobility has been
observed between different strains6,35,36. However, differences have
also been observed between different rat strains, with Wistar-Kyoto
rats showing significantly more immobility time than Sprague-
Dawley rats, coupled with reduced climbing behavior37. Similar
differences in immobility time have been observed within inbred
strains, such as Fawn-Hooded rats, which differed in their alcohol
intake38 and between the Flinders Sensitive Line and the Flinders
Resistant Line39. Moreover, the swim test has been used by research-
ers in order to breed two separate lines, namely swim-test-susceptible
and swim-test-resistant rats. After an uncontrollable shock pro-
cedure, rats that showed a large decrease in climbing behavior
90 min after stress were designated as swim-test susceptible, whereas
those that did not differ from nonshocked controls were designated
as swim-test resistant40. Notably, animals from the 12th to 15th
generations were shown to have selective differences in response
to antidepressant administration. Specifically, antidepressant
administration prevented the stress-induced decrease in climbing
behavior in the susceptible line, whereas no effect of treatment was
observed in the resistant line41.
Modified FST
The rat FST originally developed by Porsolt et al.7 has proven highly
valuable for assessing the antidepressant-like effects of the majority of
currently available antidepressants14,17,18. However, the major concern
with the traditional FST is its unreliable detection of antidepressant-
like effects of selective serotonin reuptake inhibitors (SSRIs)13,14,17.
This led Lucki and co-workers to alter specific parameters of the test
in order to increase the reliable detection of SSRIs13,34. The main alter-
ations consisted of increasing the water depth from 15–18 cm in the
original to a depth of 30 cm in the modified FST and moving from a
cumulative timing measure to a time-sampling technique wherein the
predominant behavior over each 5-s period of the 300-s test is rated.
These alterations enabled the dissection of escape-directed behaviors
into two distinct categories, namely (i) climbing, with vertical move-
ment of the forepaws; and (ii) swimming, with horizontal movement
throughout the swim chamber. The adoption of a time-sampling
technique enabled a convenient method for quantifying and distin-
guishing the two active behaviors from immobility while not differ-
ing from physically timing the duration of the individual behaviors.
In addition, these modifications decreased immobility time, as the
rats cannot stabilize themselves with their tails on the bottom of the
cylinder. Moreover, by using pharmacological and lesion studies it
emerged that the active behaviors are predominantly under the con-
trol of different neurotransmitter systems. Thus, catecholaminergic
antidepressants selectively increase climbing behavior, whereas sero-
tonergic agents selectively increase swimming behavior13,14,17,34. Thus,
the modified FST has the additional benefit of determining whether a
novel pharmacological agent predominantly activates either of these
neurotransmitter systems. For example, it has been shown that the
antidepressant-like properties of GABAB receptor antagonists are pre-
dominantly mediated via the serotonergic system, as they increase
swimming behavior, and that such behavior is attenuated by subse-
quent central 5-HT depletion16.
MATERIALS
REAGENTS
Test and control substances (see REAGENT SETUP)
Rats, n = 8–12 per group. Sprague-Dawley rats are the most widely used
and validated strain. Body weight should be consistent within experiments.
Reliable data have been generated with rats of 275–450 g ! CAUTION Experi-
menters must comply with national and institutional guidelines concerning
animal experimentation.
EQUIPMENT
Swim cylinders (usually between 1 and 4): chromatography chambers are a
useful source of suitable off-the-shelf glass cylinders
•
•
•
Thermometer (to check water temperature)
Towels (to dry animals)
Stopwatch (to time the swim trial)
Video camera mounted on a tripod and connected to a recording device for sub-
sequent analysis (the results obtained are much more accurate when scored from
recordings than from manual real-time scoring; recordings also allow re-analysis)
REAGENT SETUP
Test and control substances The only reagents required to perform the
FST are the test substances and the vehicle solution required to dissolve them
(e.g., 0.9% (wt/vol) saline solution). If you are performing the FST for the
•
•
•
•
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NATURE PROTOCOLS | VOL.7 NO.6 | 2012 | 1011
first time, it is necessary to include an antidepressant as a positive control
(e.g., imipramine or fluoxetine).
EQUIPMENT SETUP
Swim cylinders These should be tall enough to fill to a depth of 30 cm,
leaving space at the top so that the rat cannot escape, and at least 20 cm in
diameter. If the cylinders are too wide, the behavior is altered, as rats could
then freely swim. In the experimental room, place the swim cylinders in an
easily accessible location so you can gently place the animals into the water
and easily empty and refill the cylinders (i.e., close to a sink). If desired, place
the swim cylinders side by side, as previous studies have shown that there
is no behavioral effect if the animals swim in isolation or next to another
cylinder. Clearly label each cylinder (as it may not be possible to distinguish
markings on the experimental animals within) either by assigning letters to
each cylinder or, more practically, by assigning numbers to the swim cylin-
der that match those of the test animal. Such a numbering system ensures
the ability to correctly identify the test subjects and also provides blinding
to their treatment. During the test exposures, place the recording device at
least 30 cm above the cylinders and focus the image to provide a sharp, clear
image of the complete diameter of the cylinders below. For an inexperienced
scorer, recording from above reduces the likelihood of categorizing hind limb
movements that are only used for buoyancy as active behaviors. However, it
is also possible to record the swim from the side. It is normally practical to
simultaneously record two swim cylinders, as this increases the output of the
test while ensuring good recordings for later analysis. However, during the
preswim for rats, because no recording occurs, up to four cylinders can be
used simultaneously unless the rats have had a prior manipulation.
Fill the swim cylinders, for both versions of the test, to 30 cm with water
of 23–25 °C, which should be checked with a thermometer. If the tempera-
ture is warmer (i.e., 30 °C) the rats merely float, whereas lower temperatures
(i.e., 15–20 °C) lead to hypothermia and cause the animal to be more active.
PROCEDURE
Habituation
1| If rats have to be ordered from a supplier, allow at least 7 d for them to acclimatize to the housing conditions after ar-
rival. Rats can be singly or group housed but this must be consistent, as housing conditions influence swim behavior.
Handle the rats daily for a few minutes, for at least 4 days, before initiating the behavioral testing.
CRITICAL STEP The strain and age of the rats also influence swim behavior and sensitivity to antidepressant treatment.
Therefore, experimental subjects should be chosen carefully (see INTRODUCTION).
Preparation for the pretest
2| Move the animals into the pretest room, which also serves as the test room. The pretest (and test) should be performed
in a different room from the storage facility or in an adjoining room. If transportation of the rats is required, they should be
left for at least 1 h after moving to minimize the arousal caused.
CRITICAL STEP The pretest must be performed at the same stage of the light/dark cycle as other experiments in the
series. This is usually the mid-to-late part of the light cycle (i.e., the afternoon for the researcher), but some studies have
been performed in the dark phase.
Pretest
3| Fill the swim cylinders to 15–18 cm (original FST) or 30 cm (modified FST) with water of the correct temperature
(see EQUIPMENT SETUP).
CRITICAL STEP The pretest usually does not have to be recorded. However, if the rats have undergone prior manipulations
(environmental, pharmacological, lesion or genetic) that could potentially alter their behavior in the initial swim test, and
therefore in the subsequent exposure, the pretest should also be recorded.
4| Place the rats individually into the swim cylinders (one to four cylinders are appropriate) and start the timer. After
15 min of swimming, remove the rats from the water, dry them with the towels and return them to their home cages. If the
rats are group housed, ensure that no post-swim rats are placed back into a home cage with animals that still have to
undergo the preswim. In such instances, a temporary dry cage with fresh bedding is recommended until the rat can be
returned to its home cage.
5| Refill the swim cylinders with fresh water to the correct depth and again ensure that the temperature is correct.
Then repeat Step 4 with additional rats.
Drug regimen
6| If desired, administer drugs by the most appropriate route for the compound(s) of interest. The standard routes for the
swim test are s.c. or i.p. injection. However, it is also possible to inject the rats via a per os route or direct administration into
the brain. If you are treating the rats with drugs, they should generally receive three injections of the drugs or appropriate
vehicle control 1, 5 and 23.5 h before the swim test. This protocol has been well established in the literature to provide a robust
response to a wide range of antidepressant compounds. This regime is preferable as it results in prolonged brain penetration of
the compounds, mimicking a state of subchronic drug exposure and thus a continuously elevated drug concentration in the rat17.
Alternatively, a two-dose regimen, one immediately after the pretest and the second 1 h before the test, has also been shown to
be effective17, but this causes peaks and troughs of drug levels in the rat, which is less akin to the human situation.
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1012 | VOL.7 NO.6 | 2012 | NATURE PROTOCOLS
CRITICAL STEP If you are just establishing the test in your laboratory, you should include positive controls, such as
fluoxetine and imipramine, to validate the test. For Sprague-Dawley rats, 20 mg kg − 1 fluoxetine and 10 mg kg − 1 imipramine
injected i.p. or s.c. are recommended as appropriate starting points.
? TROUBLESHOOTING
Swim test
7| Twenty-four hours after the pre-test, fill the swim cylinders to the required depth with water of the correct temperature
(see EQUIPMENT SETUP). Ensure that the camera is placed directly above or to the side of the swim cylinders and that
the image produced is sharp and clear enough to allow you to distinguish the individual behaviors.
CRITICAL STEP By placing the camera above the swim cylinder, the hind limbs cannot be observed when providing
buoyancy, which could otherwise mislead the rater into scoring additional active behaviors.
8| Before placing the rats into the cylinder, begin the video recording, as it is crucial to obtain the full 5-min swim
duration. Place the rats into the swim cylinders and start the timer. After 5 min, stop recording, remove the rats from the
water, dry them with towels and then return them to their home cages.
CRITICAL STEP It is crucial that you start recording before placing a rat into the cylinder in order to capture its initial
behaviors.
CRITICAL STEP You must be sure to record the rat’s behavior for a full 5 min; otherwise the animal will have to be
excluded from the analysis.
9| Empty the cylinders and refill them with fresh water of the appropriate temperature to the correct depth and repeat
Step 8 for additional rats.
Analysis of swim test
10| Analyze the results obtained after either FST analysis (option A) or modified FST analysis (option B). In the original
version of the FST, only immobility time is traditionally measured (option A). Immobility consists of the rat floating in the
water without struggling and only making movements necessary to keep its head above water (Supplementary Video 1).
There are three predominant behaviors in the modified FST: immobility, swimming and climbing. Swimming consists of the
rat making horizontal movements throughout the swim cylinder, which also includes crossing into another quadrant
(Supplementary Video 2). Climbing comprises upward-directed movement of the forepaws, usually against the side of the
swim cylinder (Supplementary Video 3). Note that a fourth behavior, diving, can also be discerned in the modified FST,
which involves the rat diving to the bottom of the swim cylinder before resurfacing. However, this behavior occurs infre-
quently and is placed within either the swimming or climbing categories in the analysis, as long as the behavior is scored
consistently. Please see Supplementary Videos 1–3 for examples of each of the behaviors.
(A) FST analysis
(i) Use a cumulative stopwatch to obtain the total time spent in an immobile posture. We recommend that a researcher
new to the FST score a small number of rats and then reassess them to check for intra-rater reliability. Only when the
scores are within 5% should analysis of the FST commence (see TROUBLESHOOTING section). It is also highly recom-
mended that only one researcher should score an individual batch of animals given inter-rater variation, which could
obscure positive results or lead to false positives. Additional measures, such as fecal boli or latency to first immobility
behavior, have also been included by some researchers. However, these measures have never been systematically vali-
dated across laboratories and compound classes, and they should not be main measures of the test but instead used to
support the basic measures.
CRITICAL STEP The rater of the test session should be blind to the treatment group being scored.
(B) Modified FST analysis
(i) Use a time-sampling technique to analyze the results by scoring the predominant behavior in each 5-s period of the
300-s test. This will provide an overall total of 60 scores. The timer should be started immediately upon the rat’s entry
into the water and used to assess the 5-s periods. The scores can be recorded on paper or by using a counter. Similarly
to the scoring of the traditional FST, a small number of rats should be assessed twice, or until the rater reliability is
within 5–10%.
CRITICAL STEP The rater of the test session should be blind to the treatment group being scored.
Data analysis
11| Analyze the groups (if three or more) using a one-way analysis of variance (ANOVA) followed by an appropriate
post hoc test (such as Bonferroni, Fisher’s LSD or Student-Newman-Keuls) for each behavior. If there are only two groups, one
can analyze each behavior with an unpaired t test. If there is more than one factor in the experimental setup, such as drug
© 2012 Nature America, Inc. All rights reserved.
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NATURE PROTOCOLS | VOL.7 NO.6 | 2012 | 1013
administration and a preswim manipulation (i.e., previous maternal separation or stress exposure), a multifactorial ANOVA
should be used followed by a post hoc test for each behavior.
CRITICAL STEP As all versions of the swim test are the result of an activity measure, it is essential that the manipulation
assessed in the test is additionally tested in a locomotor paradigm (i.e., activity in a novel environment such as an
open-field or novel arena17). The assessment of locomotor activity is best conducted alongside the FST using the same
treatment protocol and different animals. If the same animals must be used, the locomotor test should be conducted first
and a crossover design should be used with regard to drug treatment, as the FST has been shown to have long-lasting
effects on behavior. If the manipulation causes an increase in general activity, any reduction of immobility observed in the
swim test cannot be confirmed as antidepressant-like activity (i.e., a false positive). Thus, stimulants, such as cocaine and
amphetamine, which are known to elevate mood, cannot definitively be associated with an antidepressant-like response in
the test because of the increased locomotion they cause. Contrastingly, if the manipulation causes a reduction in general
activity and a concurrent increased immobility time in the swim test, this cannot be attributed to depressive-like behavior.
However, such exclusions on the basis of locomotion are rare and may even be seen as unwanted effects of an antidepressant
in humans. Generally speaking, acute administration of the current clinically available antidepressants causes an increase
in active behaviors in the FST coupled with a reduction in locomotor activity. Thus, provided the data from the general
locomotor activity and FST are in opposing directions, then the behavior observed in the FST can be considered valid.
? TROUBLESHOOTING
The traditional versions of the swim test, particularly in rats, can be unreliable in the detection of SSRIs. Therefore, if you
are establishing the test in your laboratory you should take care when selecting the strain and dose you use. If this contin-
ues to be a problem, you should try to switch to the modified FST. Similarly, if you are finding intrarating reliability to be a
problem, switching to the modified FST may be helpful, as by scoring only the predominant behavior in each 5-s interval, the
variation can be reduced.
● TIMING
Step 1 (day 0): if animals are ordered from a supplier, they arrive and are rehoused in single cages or into groups of 2–4;
leave them for at least 7 d to habituate to the conditions. Next (days 7–11), handle the animals for 4 d to reduce the stress
of the injections
Steps 2–5 (day 12): perform the pretest
Step 6 (days 12 and 13): if desired, administer drugs
Steps 7–9 (day 13): perform the swim test
Steps 10 and 11 (day 13 onward): perform the analysis of the swim test
ANTICIPATED RESULTS
Depending on the rat strain, such as Sprague-Dawley, you should observe that vehicle-treated groups spend half the time, or
roughly 25–30 scores, in the modified FST immobile, and approximately 180–210 s of immobility time in the traditional FST.
If a manipulation is expected to be an antidepressant it should reduce immobility time in all versions of the FST, and vice
versa for a manipulation predicted to be pro-depressant. In the modified FST, it has been well established that manipulations
acting via the catecholaminergic systems, such as imipramine and DSP4 lesions, selectively alter climbing behavior (increase
and decrease, respectively)42. Contrastingly, manipulations of the serotonergic system, such as fluoxetine and the trytophan
hydroxylase inhibitor parachlorophenylalanine, selectively alter swimming behavior (increase and decrease, respectively)16.
Supplementary Data 1 gives an example of typical raw data and how this can be interpreted. For more information with
regard to anticipated results, we refer the interested reader to two excellent reviews summarizing findings from multiple
laboratories: see refs. 17,18.
Note: Supplementary information is available in the online version of the paper.
AUTHOR CONTRIBUTIONS D.A.S. and J.F.C. contributed equally to the
manuscript.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial
interests.
Published online at http://www.nature.com/doi:10.1038/nprot.2012.044.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html.
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