Knowledge Base

4 The Start-Stop trial rule

The Start-Stop trial rule is displayed on your screen when you create or open Trial Control Settings. It can be recognized by the first box with a green circle: With this rule, you control the start and stop of data acquisition (tracking). You can only modify the initial Start-Stop trial rule. You cannot: Delete the Start-Stop trial rule, including the Rule Begin, Rule End, Start track and Stop track boxes. Define an additional Start-Stop trial rule.

the default start-stop trial rule

The default Start-Stop trial rule is a sequence of six boxes (but see a few exceptions, page 48): Rule Begin - Start-Stop trial. Activated when you start the trial (from the Acquisition menu, select Start Trial, or click the Start Trial button, or press Ctrl+F5). Once you start the trial, control passes to the next box. Condition - In Zone - Cumulative duration >= 1 s. This is the default Start track condition. It is fulfilled when center point of the subject (or of any subjects, in the case of an arena with multiple subjects) has been detected in the arena for 1 second after you started the trial. If you start the trial and the animal is not detected yet, the program waits until it detects the animal for 1 second, then it starts tracking.

Figure 15 The default Start-Stop trial rule is a sequence of six boxes:

Action - Start track. Activated when the condition on its left side is met. Once this box is activated, data recording (tracking) starts. If the condition placed between the Start-Stop trial box and this box is not met immediately, tracking starts later than the time you start the trial. Condition - Time - Infinite time (condition never met). This is the default Stop track condition. This condition is never met. The trial stops when you give the Stop command or the time exceeds the maximum trial duration (when this has been set). important Always keep a Condition box that determines when the track is stopped right before the Stop track Action box, otherwise the trail stops immediately after you start the trial. Action - Stop track. Marks the end of all tracks (and trial). Rule End - Start-Stop trial. This box is just the delimiter of the rule, it does not take any action.

The Start-stop trial rule in special cases

Multiple arenas. When working with multiple arenas, the rule is applied separately for each arena. This means that tracking can start at different times in different arenas in the same trial, depending on when the condition placed immediately before the Start track box is met in a specific arena. See also a note about multiple open fields on page 52. DanioVision and Activity analysis. If you selected Activity analysis in the Experiment Settings, or created a DanioVision experiment, there is no box between the Start-stop trial and the Start track box. This means that tracking starts immediately when you click the Start trial button. For more information on Activity analysis, see Activity analysis in the EthoVision XT Help, or the DanioVision DVOC-0041 - Reference Manual. Behavior recognition. If you selected Behavior recognition in the Experiment Settings the default Trial control rule contains a Time condition box set to 3 seconds. This means that tracking starts 3 seconds after starting the trial. This is done because the Behavior recognition algorithm needs 3 seconds of video images to make the first classification of behavior. With this condition you make sure that your track contains Behavior recognition data since the first sample. If you selected both Activity analysis and Behavior recognition in the Experiment Settings, the Trial Control Rule is the same as when you selected Behavior recognition only. For more information on Behavior recognition, see Behavior recognition in the EthoVision XT Help.

An important distinction: Trial vs. track

Trial. A Trial can be viewed as a container for the data collected in one recording session. It starts when you give the Start command in acquisition and stops when the tracks for all arenas and subjects have stopped. Track. A Track corresponds to the actual recording of a subject's position and behavior. The start of a track may or may not coincide with the start of the trial. This depends on your Trial Control Settings. If you use the default Trial Control Settings, the track starts 1 second after the animal has been detected in the arena and stops when you stop the trial. A Trial may contain one or more tracks. For example, if you track two subjects simultaneously, each trial includes two tracks, one per subject. Similarly, if your setup contains four arenas with two subjects each, each trial includes 4 arenas x 2 subjects = 8 tracks. In a multiple-arena setup, the end of a track does not necessarily mean the end of the trial. The trial ends when all tracks come to an end.

customize the start-stop trial rule

Modify the Start track condition

The default Start track condition is an In zone condition. To modify that condition, click the Settings button. In the window that appears, click Settings and specify the zone in which the animal should/should not be in order for the program to start tracking. (for details on In zone conditions, see page 86). To use another condition (for example: start recording exactly 1 minute after starting the trial), delete first the current condition (click that box and press Delete) and insert the new one. For an overview of conditions, see page 26 and page 67. To start recording as soon as you start the trial, delete the Start track condition. Click the box immediately before the Start track box and press Delete.

Modify the Stop track condition

The default Stop track condition is a Time condition. To modify that condition, click the Settings button, and choose the option you require (for details on Time conditions, see page 67). To use another condition, delete first the current condition (click that box and press Delete) and insert the new one. For an overview of conditions, see page 26 and page 67. If you want to stop tracking when a specific time has elapsed, see page 21. important Keep at least one condition between Start track and Stop track. If you do not do this, tracking stops immediately after tracking starts, resulting in no data. important If you have inserted Condition boxes based on Activity in your Trial Control rule and then deselect Activity analysis in the Experiment settings (see Activity analysis in the EthoVision XT Help), the Condition boxes based on Activity are removed from your sequence and the connecting arrows are removed. This makes the rule invalid. Redesign your Trial Control rule and connect the arrows between the boxes.

Call a sub-rule

Sub-rules are subroutines of the Start-Stop trial rule (see page 55). You can program EthoVision XT in such a way that a subset of Trial control actions and conditions is called from a specific point in the Start-Stop trial rule. When this subset of instructions is finished (or repeated a number of times), the program resumes following the instructions of the Start-Stop trial rule. 1. Define a sub-rule (for details see page 55). 2. In the Components pane, under Structures, click the button next to Sub-rule reference. Enter a name for the reference, and select the sub-rule you have defined in step 1 above. 3. Insert the reference box in the correct place in the Start-Stop trial rule. For more information on sub-rules, see page 55. For more information on sub-rule references, see page 57. You cannot define a sub-rule reference without defining the sub-rule first.

examples of start-stop trial rules

Start data recording at a specific time

You want to start recording at a time you are not in the lab, for example at 23:00 h. Delete the default Start track condition (see page 49). Define a Time condition (see page 67). Select At clock time and enter 23:00:00. Click OK and place the resulting box before the Start track box. Before leaving the lab, click the Start trial button to start the trial. The program waits till 23:00 to start data recording. For a similar example, see Examples of Start-Stop trial rules in the EthoVision XT Help.

Stop data recording after the maximum time has elapsed

Click Settings in the Condition box immediately before the Stop track box. Select After and enter the maximum time. note Instead of using a Time condition, you can also use the Maximum trial duration option (see page 21).

Water maze: Stop the trial when the animal has found the platform

In the Arena Settings, make sure that the platform has been defined as zone. In the Trial Control Settings, delete the default Stop track condition (see page 49). Next, define a In Zone condition (see page 86). If you want the program to stop recording as soon as the animal is over the platform, select Frequency as Statistic and choose >= 1. Click Settings and select the platform zone. Sometimes the animal swims over the platform, but it does not stop there. In such cases the program would stop recording while the animal has not "found" the platform. Instead of selecting Frequency, choose Current duration and the minimum time the animal must stay on the platform (for example, 3 seconds). Click Settings and select the platform zone. Click OK and place the resulting box before the Stop track box. Water maze: Stop the trial either when the rat has found the platform, or when it has been

swimming in the water maze for 60 seconds

The Arena Settings and the condition In platform zone are similar to those in the example above. The condition subject swimming for 60 s can be translated to Time >= 60 s. The track stops when either condition is met. The two conditions are combined with OR logic (see Figure 16). This solution results in tracks of different duration: less than 60 s for the animals that found the platform, and 60 s for the others. tip Instead of two Condition boxes in the example above, you can also define the In zone condition box and set a Maximum Trial duration (see page 21). Multiple open fields (multiple arenas): Starting tracking when the subject is released in the open field. Tracking starts independently in each open field. In this setup, four open fields are treated as separate arenas. You want to start acquisition when the animal is detected in the open field independent of what happens in other arenas. This can be achieved by using the default Start-Stop trial rule. As soon as an the subject is detected in an arena, tracking starts for that arena, not the others. This way you do not have to release all the animals at the same time. Conditioning test: Stop the trial when the conditioning task has been repeated N times. For example, when the animal has obtained 25 rewards. 1. Create a new sub-rule (see page 55) containing the instructions for the testing session. Sub-rule Begin Condition Is subject on top of the shelter? Action Drop a pellet Sub-rule End. Figure 16 Example of a Start-Stop trial rule for a water maze. The trial stops when the animal has been in the platform zone for at least 3 s without break, or the time since the start of tracking is 60 s. A - In zone condition that specifies that the animal must be for at least 3 seconds over the Platform zone. Select Current duration >=3s. B - Time condition that specifies a time of 60 s since the track started. C - "Any" operator box. Since the sub-rule is restarted immediately after the pellet has been dropped, it is possible that the animal is still on the top of the shelter, and a new pellet is dropped before the animal eats the previous one. Therefore, you must make sure that the sub- rule ends when the animal has visited the reward zone, so the sub-rule can be repeated from that moment onward. You can do this by defining a condition "In Feeder zone" that is met when the animal has been in the Feeder zone for at least 1 s without break (Current duration >=1 s). Place this box to the left of Rule End. 2. Create a Reference to the sub-rule (see page 57) and specify to have the sub-rule repeated 25 times. Place the resulting box at the left side of the Stop track box. Eight-arm radial maze: Stop the trial when the subject has been in four arms within 10 minutes. Combine eight conditions, one for each arm. At least four conditions must be met, no matter which arm the animal visits. 1. Create an In zone condition (see page 86) and specify that the Frequency for Arm 1 must be >=1. That is, the animal must have visited Arm 1 at least once. Do the same for each of the other arms. 2. Connect the resulting eight condition boxes in parallel using the N of All operator (see Figure 17). 3. Set the Maximum trial duration (see page 21) to 10 minutes to stop tracking in the case the animal fails to visit four arms in the meantime. For more information on operators of type N of All, see page 34. important The frequency of In zone can become high very easily when the animal walks along the edge of a zone. This may result in the condition being met sooner than expected. If that happens, increase the Zone exit threshold. If that does not help, open the Detection settings and click Advanced, then under Smoothing set Track noise reduction to On. This smooths the track. Figure 17 A Trial Control sequence for an eight-arm radial maze. The trial must stop when the animal has visited four of the arms at least once. zone for that arm is greater than or equal to 1. A - Operator that checks that at least four of the eight conditions are met. B- Stop track box. When four conditions are met, the trial is stopped.

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Source: EthoVision XT 17 - Trial and Hardware Control Reference Manual