Knowledge Base

A Conditioning Test in EthoVision (Continued)

1. In the Sub-rule window, type 'Anxiety Test' in the Sub-rule name field. Optionally, enter a comment and click OK.

2. Create an In Zone Condition for when the animal enters the Spot Light Zone:

   1. In the Components pane, double-click the In zone Condition or click the button next to it.
   2. In the In zone Condition window, type 'Inside LightSpot' in the Condition name field.

   3. In the Condition is met when: group, from the Statistic list, select Frequency.

      Next, click the Settings button, select zone Spot Light from In the following zones, and select When in any of the selected zones from the list.

      In the From the following body points group, select both Nose-point and Center-point. Make sure to select All selected points from the list.

   4. Click OK.
   5. Insert the Condition box into the Sub-rule sequence.

3. Create a Hardware Action (White Light on) for when the previous Condition is met:

   1. In the Components pane, under Actions - Hardware, double-click Top Unit (standard) or click the button next to it. If a third-party device has been connected, under Actions - Hardware double-click Custom Hardware.
   2. Next to Action name, type "White Light on".
   3. Next to Action to perform, select White spot on from the list and click OK.
   4. Insert the Action box into the Sub-rule sequence. If a third-party device has been connected, select an action from the Custom Hardware.

4. Create an In Zone Condition for when the animal leaves the Zone LightSpot:

   1. In the Components pane, double-click the In zone Condition or click the button next to it.
   2. In the In zone Condition window, type 'Outside LightSpot' in the Condition name field.

   3. In the Condition is met when: group, from the Statistic list, select Frequency.

      Next, click the Settings button, select zone Spot Light from In the following zones and select When not in any of the zones.

      In the From the following body points group, select both Nose-point and Center-point. Make sure to select All selected points from the list at the bottom.

   4. Click OK.
   5. Insert the Condition box into the Sub-rule sequence.

5. Create a Hardware Action (White spotlight off) for when the previous Condition is met:

   1. In the Components pane, under Actions - Hardware, double-click Top Unit (standard) or click the button next to it.
   2. Next to Action name, type White Light off.
   3. Next to Action to perform, select White spot off from the list and click OK.
   4. Insert the Action box into the Sub-rule sequence. If a third-party device has been connected, select an action from the Custom Hardware.

The Sub-rule Anxiety Test is now completed.

Sub-rule Reference 'Start Anxiety Test'

8. To create a Reference in the main sequence to the Sub-rule LightSpot:

   1. In the Components pane, under Structures, double-click Sub-rule reference or click the button next to it.
   2. Next to Reference name, type Start Anxiety Test.
   3. Next to Reference to sub-rule, select Anxiety Test.
   4. Next to Stop Conditions, select Repeat per start condition. Select for a number of ... times and set the value accordingly.
   5. Click OK and insert the Reference box into the main sequence.

Condition 'Stop Delay'

9. To insert a 5-second delay before the trial is stopped:

   1. In the Components pane, under Conditions, double-click the Time Condition or click the button next to it.
   2. If the Add a condition window appears, it means there is at least one condition of the same type in your experiment. You are asked to choose between creating a new condition or re-using an existing one. Choose the option you require and click OK. If this window does not appear, skip this step.
   3. In the Time condition window, type 'After 5 seconds' in the Condition name field. In the Condition is met: group, select After a time of 5 secs. Optionally, enter a comment and click OK.
   4. Insert the Condition box into the main sequence.

For more information and examples:

• About PhenoTyper: PhenoTyper - EthoVision XT 18 - Reference Manual.
• About subrules, conditions and actions: EthoVision XT 18 - Trial and Hardware Control - Reference Manual.

Detection Settings

Choose Setup > Detection Settings > open Detection Settings 1.

1. Check in the Video Section that the sample rate is set to:

   • Rats: 5 samples/second.
   • Mice: 12.5 samples/second.
   • Nose-tail tracking: 25–30 samples/second.

2. Under Method, choose your detection method and the contrast with the background.

   If you selected to use the Deep learning technique to track the subject's nose, next to Deep learning click Define and select a box around the subject. Make sure that the box includes the subject's nose and does not include objects of the same color as the subject. For details, see the EthoVision XT Help.

3. If you selected Activity analysis in the Experiment settings, open the Activity section in the Detection Settings pane. 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 so that when the animal is completely still, those pixels should be almost completely absent. Leave the Compression artifacts filter off if you track live.

Trial List

Enter your independent variables such as rat ID, first or second phase of the experiment, treatment (drug vs. control), dose, duration of treatment, name of the experimenter, etc. You can pre-define all your trials here, 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.

To carry out batch acquisition, plan your trials in the Trial list. Optionally select Arena Settings, Trial Control Settings, and Detection Settings.

Source: EthoVision XT 18 - Application Manual, Noldus Information Technology