Knowledge Base

Introduction

The Porsolt Forced Swim Test (Porsolt et al., 1977; Hedou 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.

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

• Castagne, 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.
• Hedou, 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.

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 cylinders 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.

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.

EthoVision XT Settings

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 correspond 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 in the Acquiring Data section). This means that you do not have to manually score the subject's behavior.

However, if you want to compare the data scored by EthoVision XT with your own ratings, define behavioral categories under Manual Scoring Settings. Next, during acquisition watch the video and score those categories during your trial. You can also edit the data after acquisition.

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.

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)

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.
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.

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).

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 minutes 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. If you need more time to put an animal in its cylinder, increase the number of seconds. This way you prevent detection of your hand or arm from starting tracking. The Start Condition works independently for each cylinder.
• Max Track Duration 5 mins. This profile is similar to the previous one, but 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: 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.

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.

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 Castagne et al. (2011). Current Protocols in Neuroscience 8.10A.1-8.10A.14.

Score Behaviors Manually

If you want to score the subject's behavior manually, define behaviors first under Manual Scoring Settings. 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 for the category that applies. As soon as the behavior of the subject changes, click the button for the corresponding category.

• 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, 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 and visualize the data.

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 calculation of Activity-based variables.

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