The forced swim test

The forced swim test uses water too, but there's no platform to find. It measures how much an animal climbs, swims, dives or floats in a container it can't escape from.

Forced swim test in eight steps

Calibrate a band around the water line, track the body rather than the tail, run, then read % time climbing, swimming, floating and diving.

Make the background behind and around the containers white for dark or hooded animals, or black for white animals; for three containers, place them in a row, close together, in the top third of the view.
Click ‘Open Field and NOR’ and leave ‘2D-tracking mode’ showing.
Click ‘Calibrate’ and select the open field maze type. The tracking area is a band around the water line: size it so the animal's highest point is in the top third when climbing, the middle third when swimming or floating, and the bottom third when diving.
Click the top-left then the bottom-right corner of the band and click ‘Save (and return)’.
Click ‘Run’ and choose ‘Single Field’ for one container or ‘3x3 Fields’ for three in a row; on the ‘Tracking’ tab move ‘Set X-Y Threshold’ until a cross follows each animal, and move ‘Set X-Y Size’ right if it jumps to the tail.
On the ‘Configuration’ tab enter ‘Max secs’; on the ‘Run a Trial’ tab click ‘Prepare to Run’, place the animals and click ‘Run’.
With three containers, nine .hvs files are made per set: move the files ending 4 to 9 to another folder before analysing.
Click ‘Analyze’, select OF and ZA and click ‘Analyze Single’ for % time climbing, diving, swimming and floating; use ‘Setup Custom’ and ‘Auto-Analyze’ for many trials.

More detail

For three containers, enter each subject then six dummy names, separated by pipes. Delete the dummy files only after a successful analysis.

The software uses the highest part of the animal within the tracking area to classify climbing, swimming/floating and diving, and separates swimming from floating by speed, so the band may be quite shallow relative to its width.

Forced swim % climbing, swimming, floating, diving

Forced swim results give the % of time the subject spent climbing, diving, swimming and floating, with total path, duration, average speed and % time moving.

More detail

‘Analyze Single’ shows these in the trial summary; for batches use ‘Setup Custom’, where HVS suggests average speed, path length, zone entries and the four % time measures. With a row of three run as a 3 x 3 grid, nine .hvs files are made per set, and files ending 4 to 9 should be moved out of the HVS folder before analysis.

Grid cells are optional for FST. You can make several CSTs with different grid sizes or measure sets.

Reading forced swim results

HVS Image gives % time climbing, diving, swimming and floating, plus speed and path. Check the tracking band and tail tracking before you interpret the percentages.

Typical control pattern: No published pattern has been confirmed for this test yet, so compare with your own control group.
Signs of impairment: No published pattern has been confirmed for this test yet, so compare with your own control group.
Signs of strong memory: No published pattern has been confirmed for this test yet, so compare with your own control group.

Set-up problems that look like results

Cross jumps to a raised tail or water droplets on the container wall: move ‘Set X-Y Size’ to the right.
Climbing, swimming and diving look misclassified: check the tracking band height, so the animal's highest point is in the top third when climbing, the middle third when swimming or floating and the bottom third when diving.
Cross will not stay with the animal at any slider position: improve lighting and camera settings for an even background, good contrast and no glare.

More detail

The software uses the highest part of the animal within the tracking area to classify climbing, swimming/floating and diving, and separates swimming from floating by speed. With three containers run as a 3 x 3 grid, only the files for the real containers are your data.

No forced swim paper is yet in the verified publications database (a classic reference has been proposed and awaits approval), so no control, impaired or enhanced pattern is given here.

Questions people ask

How do I set the tracking area for the forced swim test?

Calibrate a band around the water line, sized so the animal's highest point sits in the middle third when swimming or floating. 1) Click 'Open Field and NOR' > 'Calibrate' > 'Open Field or Zone Analysis'. 2) Use the full width of the cylinder. 3) Choose a height where climbing puts the highest point in the top third and diving in the bottom third. 4) Click top-left and bottom-right, then 'Save (and return)'.

More detail

For FST click 'Open Field and NOR' and leave '2D-tracking mode' showing. For a single container the tracking area is the band around the water line, not the whole container. Choose its height so that the animal's highest point (nose or back) is in the top third when climbing or seeking escape upwards, the middle third when swimming or floating, and the bottom third when diving. Enter user name and the height and width, tick black only for light animals on a dark background, click top-left and bottom-right, then 'Save (and return)'. The whole background, including around and behind containers, must be white for dark or hooded animals or black for white animals.

Going deeper

The software uses the highest part of the animal within the tracking area to classify climbing, swimming/floating and diving, and separates swimming from floating by speed; body parts beyond the top or bottom of the area do not matter, so the area may be quite shallow relative to its width. For three containers in a row: place them close together in the top third of the view, mark one container's tracking band, multiply its height by three and the mid-gap-to-mid-gap width by three, mark the resulting bottom-right point, then click the first container's top-left and that point. The rectangular-arena rule applies: click top-left first, then bottom-right.

How do I run and track forced swim trials, including three containers at once?

Track the body, not the tail, by moving 'Set X-Y Size' right; for three containers use '3x3 Fields' with six dummy names. 1) 'Run' > 'Maze Type' tab > 'Open Field or Zone Analysis' > 'Single Field' (or '3x3 Fields' for three in a row). 2) On the 'Tracking' tab set 'Set X-Y Threshold' until a cross follows each animal. 3) Enter subjects like subject 1|subject 2|subject 3|-|-|-|-|-|- and the same for descriptors. 4) 'Prepare to Run', then 'Run'.

More detail

From 'Run', on the 'Maze Type' tab select 'Open Field or Zone Analysis', choose 'Single Field' for one container or the grid with the right number of columns ('3x3 Fields' for three in a row), and open the 'Tracking' tab. With trial lighting and an animal in each container, set 'Set X-Y Threshold' until a green or red cross tracks each animal. It is important not to track the tail: if the cross jumps to a raised tail or water droplets on the container wall, move 'Set X-Y Size' right. On the 'Configuration' tab set 'Max secs' and 'Single Trial' or 'Series of Trials'. For three containers enter each subject followed by six dummy names separated by pipes, e.g. subject 1|subject 2|subject 3|-|-|-|-|-|-, and likewise for descriptors (in a .txp: ,,subject 1|subject 2|subject 3|-|-|-|-|-|-,group A|group B|group C|-|-|-|-|-|-). Click 'Prepare to Run', place the animals and click 'Run'.

Going deeper

If the cross will not stay with the animal at any slider position, improve lighting and camera settings for even background, good contrast and no glare. Untick 'Record' when tracking pre-recorded videos.

How do I analyse forced swim results and get % time climbing, diving, swimming and floating?

Analyse in OF and ZA; the forced swim measures appear in the trial summary and can be exported via 'Setup Custom'. 1) If you ran three in a row, first move .hvs files ending 4 to 9 out of the HVS folder. 2) 'Analyze' > OF and ZA > click a trial > 'Analyze Single'. 3) For many trials, 'Setup Custom' > tick Field Variables (speed, path, zone entries, % climbing, diving, swimming, floating) > 'Save Spec' > 'Auto-Analyze'.

More detail

With a row of three run as a 3 x 3 grid, nine .hvs files are made per set; with the containers at the top of the view, files ending 1 to 3 are your data and files ending 4 to 9 should be moved out of C:\HVS Image 2027\Users\[PC username]\Data\Open Field Test\HVS (delete them only after successful analysis). Then click 'Open Field and NOR' > 'Analyze', select OF and ZA, keep Video Height/Width at 480/640 for live trials, and click a trial. 'Analyze Single' shows total path, duration, average speed, % time moving, zone measures and % time climbing, diving, swimming and floating; Print or Export gives a text file. For batches use 'Setup Custom', ignore 'Maze Variables', and in 'Field Variables' HVS suggests average speed, path length, zone entries and the four % time measures; 'Save Spec', select the CST file, select trials and 'Auto-Analyze'. CST files are in …\Data\Open Field Test\CST.

Going deeper

Grid cells are optional for FST. CST files are comma-separated: save a copy as .csv or import into Excel. You can make several CSTs with different grid sizes or measure sets.

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