Knowledge Base

The Porsolt Forced Swim Test

Introduction

the porsolt forced swim test

The Porsolt Forced Swim Test (Porsolt et al., 1977; Hédou et al., 2001; Juszczak et al., 2008) is the most commonly used test for assessment of depression in animal models. In the Porsolt test, a state of depression is induced by forcing a rodent to swim in a narrow cylinder or water tank from which it cannot escape. The procedure is generally divided into a pre-test session lasting 15 minutes, followed 24 hours later by a 5-minute test session. After a period of vigorous activity in the pre-test session, the animal adopts a characteristic immobile posture. Twenty-four hours later, after administration of antidepressants, the effect on immobility can be investigated by measuring the time of being immobile in the test session and comparing this with control animals. Figure 9.1 A Porsolt forced swim test carried out in EthoVision XT.

the sample experiment

To see how a Porsolt forced swim test is carried out in EthoVision XT,

see the sample experiment Porsolt Forced Swim test XT175 on the

downloads section of the Noldus website (see my.noldus.com). Download this file and save it on your computer. Next, in EthoVision XT,

select File, Restore Backup and select this file. See also the document

Description of sample experiments of EthoVision XT.pdf for more

information.

references

Castagné, V., Moser, P., Roux, S., and Porsolt, R.D. (2011) Rodent Models of Depression: Forced Swim and Tail Suspension Behavioral Despair Tests in Rats and Mice. Current Protocols in Neuroscience 8.10A.1- 8.10A.14. Juszczak, G.R., Lisowski, P., Sliwa, A.T., and Swiergiel, A.H. (2008). Computer assisted video analysis of swimming performance in a forced swim test: Simultaneous assessment of duration of immobility and swimming style in mice selected for high and low swim-stress induced analgesia, Physiology and Behavior 95: 400-407. Hédou, G., Pryce, C., Di Iorio, L., Heidbreder, C.A., and Feldon, J. (2001). An automated analysis of rat behavior in the forced swim test, Pharmacology, Biochemistry and Behavior 70: 65-76. Porsolt, R.D., Bertin, A., and Jalfre, M. (1977). Behavioral despair in mice: a primary screening test for antidepressants. Archives Internationales de Pharmacodynamie et de Therapie 229: 327 - 36.

EthoVision XT and the Porsolt forced swim test

For a list of selected publications, see the results of Google Scholar.

Physical setup

The following suggestions are meant to optimize the quality of tracking.

Camera view

The camera should have a good, frontal view of the entire region the animals can be in. The complete body of the animal should be visible.

Number of animals

You can test two or more animals simultaneously, when the Porsolt cylinder fit in the same camera view. For tracking multiple animals you need the Multiple Arenas add-on module.

Contrast

Try to create as much contrast as possible between the animal and the background. This can be done by placing the cylinder or tank against a contrasting, non-reflecting background (so, a matt black background for white animals and vice versa). You can also use back-lighting to create maximum contrast. In this case, make sure you place a screen or curtain between the lights and the cylinders to avoid reflections.

Figure 9.2 Backlighting allows to improve contrast between animal

and background, and minimize reflections.

Water surface

You can reduce the contrast due to the water surface by placing the cylinders in a bigger tank with the water surface of the tank above those of the cylinders. Figure 9.3 Place the Porsolt cylinder in a water tank to remove or minimize the image of the water surface inside the cylinder.

The Porsolt Forced Swim test in EthoVision XT

Create an experiment. For details, see Chapter 1 of this manual, or in

EthoVision XT press F1 and see Setup an Experiment in the EthoVision

XT Help.

experiment settings

Choose Setup > Experiment Settings.

Under Video Source, and Tracked Features, make sure that the options

selected corresponds to your needs. To adjust the camera settings, click the video icon in the camera row.

Under Analysis options select Activity analysis. Click the video button

next to it and watch the video tutorial to learn about Activity analysis. Activity is a useful feature that helps you detect changes in the movement patterns of the subject.

manual scoring settings

EthoVision determines the behavior of your subjects automatically on

the basis of the intensity of the subject's movement (see determining

mobility thresholds on page 169). This means that you do not have to

manually score the subject's behavior manually. However, if you want to compare the data scored by EthoVision XT with

your own ratings, define behavioral categories under Manual Scoring

Settings (see below). Next, during acquisition watch the video and

score those categories during your trial. You can also edit the data after acquisition.

Procedure

Choose Setup > Manual Scoring Settings. 1. To add a behavior, click the Add Behavior button. Enter a name for

that behavior (for example, Struggling) and specify Mutually

exclusive as Behavior type. Specify the name of the behavior group

(for example, Behavior Categories). 2. Add the remaining behaviors to the same group.

Notes

We advise you to create three categories for scoring the animal's mobility levels, which correspond to the three possible states of the Mobility dependent variable in EthoVision XT: - Immobile (corresponding to EthoVision XT's Immobile state); - Swimming (corresponding to Mobile); - Struggling/Climbing (corresponding to Highly Mobile) See score behaviors manually on page 168.

arena settings

1. Choose Setup > Arena Settings > Open Arena Settings 1.
2. Click 2. Select Shape and Draw Arena and check that the pre-defined arenas have the correct shape and size. If necessary, adjust the contour of the arena to fit the cylinders.

important If you work with multiple cylinders and use Activity

analysis to quantify the animals' behavior, make sure that the

arenas have the same size (you can check this under Shape Size and

Position in the Arena Settings window). 3. Click 3. Select Shape and Draw Zones. Check that the zones (if you used a Zone template) have the correct shape and size. If necessary, draw additional zone groups/zones. For details, see Arena Settings in the EthoVision XT Help.

trial control settings

Choose Setup > Trial Control Settings > Open one of the following:

Default. In this profile, tracking starts 1 second after the animal has

been detected in the arena. Tracking stops manually (for example when you click the Stop trial button).

Max Track Duration 15 mins. In this profile, tracking starts 1 second

after the animal has been detected in the arena. Tracking stops automatically after 15 mins or when the animal has been in the Diving zone for over 1 second. You can use these Trial Control Settings for the pre-test session (See protocol on page 168). If you need more time to put an animal in its cylinder, increase the number of seconds. This way you prevent that detection of your

hand or arm starts tracking. The Start Condition works

independently for each cylinder. Figure 9.4 Example of Arena Settings for the Porsolt Swim test with two arenas.

Max Track Duration 5 mins - This profile is similar to the previous

one, but now the maximum tracking duration is 5 minutes. You can use these Trial Control Settings for the test session. When you work with multiple arenas, trial control is applied to each arena separately. Therefore, tracking may start at different times in different arenas, depending on when the animal is released.

detection settings

Important note

It is important for stable detection, that the extremities of the animal (paws and tail) are either always included with the tracked body or always excluded from it. If the paws and tail are temporarily excluded and then included again in the detected subject during data

acquisition, these changes affect the variables Mobility and

Movement, while the animal might not actually be mobile or moving. On the other hand, the variable Activity is not affected by such changes, because it is based on the image change in the whole arena, not the change in the detected subject.

Detection Settings

1. Choose Setup > Detection Settings > Detection Settings 1.
2. Under Video, choose a Sample rate of 5 (for rats) or 12.5 samples/ second (for mice) or higher.
3. Click Advanced.
4. Under Method, choose Dynamic subtraction.

Under Detection, check you selected the correct contrast. Move the

slider to define the animal's contrast. The animal's body must be fully detected in all parts of the arena and the noise must be

minimal. Set the Current frame weight slider to a value between 1

and 5 to adjust the number of past images that contribute to the reference image. 5. If the limbs and the tail are not included in the detected body, and

you want to include them, under Subject Contour select the Dilation

filter, then the second Erosion filter. Leave the first Erosion filter to

zero. Adjust Dilation and Erosion to achieve stable detection. 6. Under Subject Size, click Edit. Define a Minimum Subject size to prevent small objects like droppings from being detected. 7. If you intend to use Activity analysis to score the behavior of the

subject automatically, click Activity. Enter the Activity threshold for

detecting a change in the pixels in the arena. Start with the default value and check the purple-colored pixels in the video image. Adjust the threshold in such a way that when the animal is immobile, those pixels are almost completely absent.

Set Compression artifacts filter to On if you work with pre-recorded

video.

trial list

Choose Setup > Trial List. Enter your independent variables such as rat ID, Session type (with values Pretest or Test), Treatment (with values Drug vs. Control), Dose, Time of drug administration, Name of the experimenter, etc. For variables which differ between subjects, select Subject as a Scope.

If you want you can pre-define all your trials here (click the Add Trials

button), or you can enter the independent variable values as you carry out the trials. You can also prepare the trials in Excel, randomize them and then paste the values into your Trial List. Furthermore, you can define a list of trials for batch acquisition. See Acquire a series of trials in the EthoVision Help. Figure 9.5 A trial list with four planned trials. Each trial includes two arenas, and therefore two subjects.

Acquiring data

protocol

Basic protocol (rats)

Pre-test trial (day 1, before drug administration). The animal is

placed in the cylinder for 15 minutes. No scoring of immobility is performed during the first session. This session is needed to acclimatize the rats to the experimental situation and to induce a stable, high level of immobility during the test session.

Test (day 2). After drug administration, the animal is placed in the

cylinder for 5 minutes. Score the duration of immobility by summing the time spent immobile; score as immobile minor movements strictly necessary to maintain the animal's head above water.

Alternate protocol (mice)

The mouse is placed in the cylinder. The trial lasts 6 minutes. Measure the latency to immobility from the start of the trial and the duration of immobility for the last 4 minutes. See Castagné et al. (2011). Current Protocols in Neuroscience 8.10A.1- 8.10A.14. See acquiring tracks on page 21.

score behaviors manually

If you want to score the subject's behavior manually, define them first

under Manual Scoring Settings (page 162). After you have started the

trial, watch the subjects in the video and in the Manual Scoring tab at

the bottom of the screen, click the button to the category that applies. As soon as the behavior of the subject changes, click the button for the corresponding category.

Notes

important If you do tracking from video, make sure you de-select

DDS in the Playback Control window.

You can also score behaviors after tracking. See Acquire Data >

Score behaviors manually in the EthoVision XT Help.

determining mobility thresholds

To find the optimal Highly mobile and Immobile thresholds, you can

run a few test trials and check in the Analysis Results and Scoring pane

(Dependent Variables tab) the values for Mobility state in relation to

the behavior the animal displays. If the Mobility states do not

correspond to the behavior of the animal, in the Analysis Results and

Scoring pane click Dependent Variables (see figure below), click the

button under Mobility state and adjust the Highly mobile threshold

and Immobile threshold.

For example, if the animal is struggling, its corresponding Mobility

state is Highly mobile. If the current Mobility score is Mobile or

Immobile, you probably need to reduce the Highly mobile threshold. Note that you can also adjust the Mobility thresholds after acquisition.

in the Analysis profile, add the Mobility state variable (see page 173),

and visualize the data.

Figure 9.6 During acquisition, you can check the values of any dependent

variable in real-time. In this example, Mobility and Mobility state have been

chosen. Mobility state has three possible states, Immobile, Mobile and Highly

mobile, depending on where the value of Mobility lies relative to some user-

defined thresholds. In Arena 1, the animal is struggling and the current value of Mobility is about 14%. In order to score struggling as "Strongly mobile", set the Highly mobile threshold to, for example, 12%.

If you do not see the Mobility state variable on your screen, click Show/

Hide on the toolbar and select Show Dependent variables, then

Mobility state and Mobility.

determining activity thresholds

In the same way as you defined thresholds for Mobility state, you can

define thresholds for Activity state. Use Activity state if Mobility state

does not give satisfying results, for instance when detecting thrashing of the animal's limbs which may not be detected properly by Mobility. Note that when you work with multiple cylinders in the same video image, Activity state gives good results when the size of the arena is the same for all cylinders. In the Arena Settings, adjust the contour of the arenas to make them similar.

Data analysis

data preparation

Track editing

Choose Acquisition > Edit Tracks. You can fix tracking errors but normally you will not need to edit your data.

Track smoothing

Choose Acquisition > Track Smoothing Profile > open MDM 0.2 cm. In

this profile, the Minimal Distance Moved filter is used (option set to

Direct, with a threshold of 2 mm). This filter filters out small

movements which result from system noise. Note that smoothing influences the track, that is, the coordinates of the center point of the subject, not the subject's detected area, and therefore does not influence the calculations of Mobility. It also has no effect on Activity analysis.

Review video and behaviors

If you have scored behaviors manually, you can review the video and if

necessary edit the data. Choose Acquisition > Score behaviors

manually. For details, see Acquire Data > Score behaviors manually in

the EthoVision XT Help.

Selecting data

Choose Analysis > Data Profile. You can select your tracks according to your independent variable values and also select parts of tracks (nesting). You can also create groups of tracks to obtain group statistics for each group. Figure 9.7 shows an example of a Data Profile to compare the animals that were treated with a drug, with those that were treated with saline.

integrated visualization

Choose Analysis > Results > Plot Integrated Data. Select a trial from the list on the tool bar. The video file and the Time Event Plot are displayed. In the Time Event Plot tool bar you can choose which profiles to use for display of the variables. Figure 9.8 shows how variables are visualized

in the sample experiment Porsolt Forced Swim Test XT175 with the

Analysis Profile Mobility_10 containing the variable Mobility. Play the video and check whether the states scored under Mobility state correspond to the behavior of the animal. If they don't, fine tune the Mobility (or Activity) thresholds in the Analysis profile (see page 173). Then, check the results of your changes again in the Integrated Visualization.

Figure 9.7 An example of data selection to compare two data sets in your

experiment.

analysis profile

Mobility analysis

In an Analysis Profile, you select the dependent variables and statistics for analysis. For the experiment made with the template for the Porsolt Test, a dependent variable Mobility is already present. Choose Analysis > Analysis Profile > Open > Mobility. 1. Double-click the row for the Mobility state variable. 2. Under Outlier filter, the Averaging interval is the number of samples

on which the running average mobility is based. With an Averaging

interval of 1 sample, sudden changes in surface area caused by

reflections are not filtered out. Figure 9.8 The Plot Integrated Data screen for the Analysis profile 'Mobility_10' in sample experiment Porsolt Forced Swim Test XT175. The plot shows the variables

Mobility (line plot) and the duration of Mobility state (colored bars) for both

animals (here indicated as Arena 1 and Arena 2). The vertical line shows the current position in the Time Event Plot and the video window displays the

corresponding frame. You can use this function to fine tune the Mobility state

thresholds and detect the behaviors you are interested in. 3. Under Threshold, set the Highly mobile and Immobile thresholds to

the values you have previously determined (see determining

mobility thresholds on page 169). 4. Under Calculate statistics for, select the states you want analyze. 5. Click the Trial Statistics tab and select Duration and Frequency. Optionally select additional statistics in the Group statistics tab. 6. Click OK.

Comparing EthoVision Mobility data with your ratings

If you have scored the animal's behavior manually, you can easily compare your data with those scored by EthoVision (Mobility). Make

sure that your analysis profile includes the three Mobility states and

the behaviors scored manually, then visualize the data. If the EthoVision data do not match your ratings at an acceptable level, adjust the Mobility threshold values in acquisition (page 169) and in analysis (see above).

An alternative to Mobility analysis

You can use Activity state if the Mobility state variable does not give

good results, for instance when detecting thrashing of the animal's limbs which may not be detected properly by Mobility state.

Activity analysis is based on the temporal change in pixel value in the

whole arena. Because it is not based on the detected animal's surface area, it is independent of tracking. 1. In the Experiment Settings, under Analysis options select Activity

analysis. Click the video button next to it and watch the video

tutorial to learn about Activity analysis. 2. In the Detection Settings, click Activity. Enter the Activity threshold for detecting a change in the pixels in the arena. Start with the default value and check the purple-colored pixels in the video image. Adjust the threshold in such a way that when the animal is immobile, those pixels are almost completely absent. 3. Run the trial. 4. In the Analysis profile add Activity and Activity state. 5. Visualize the data (Plot Integrated Data). There you see the scored

Activity states and the subject in the video (see Figure 9.8 for an

example with Mobility states). Adjust the Activity state settings in such a way that the states scored by EthoVision XT match the behavior of the subject at that time.

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Source: EthoVision XT 17.5 Application Manual, The Porsolt Forced Swim Test