Knowledge Base

Introduction

EthoVision XT can be used in learning tests, such as the water maze test and the conditioning tests.

The Trial and Hardware Control module of EthoVision XT can be used to operate hardware devices to be used in both classical and operant conditioning tests. In the latter, an action of the animal produces a result, for example, pressing a lever results in the food dispenser dropping a food pellet. This and other hardware actions (such as turning on a light when the animal enters the shelter, or closing the door of a radial maze when the animal has exited that arm) can be operated through the Trial and Hardware Control in EthoVision XT.

Learning can be demonstrated by a change in the frequency of the animal's action over time. For example, when the animal is presented with an aversive stimulus every time it performs a certain action, the performance of the action will likely decrease over time because the animal tries to avoid the negative stimulus.

To be able to operate hardware devices with EthoVision XT you need:

• The Trial and Hardware Control module. This module allows you to control hardware devices from within EthoVision XT.
• The USB-IO Box. This is the device that connects the EthoVision XT PC with external hardware devices such as the sound and light devices of PhenoTyper or a third-party hardware device.

This document describes how to set up EthoVision XT using Trial and Hardware Control in an operant conditioning test in PhenoTyper.

PhenoTyper

PhenoTyper is an instrumented cage to measure and test the behavior of laboratory rodents. See www.noldus.com/animal-behavior-research/products/phenotyper for more information on PhenoTyper.

If you want to use EthoVision XT to operate a third-party hardware device, please contact Noldus Information Technology at www.noldus.com.

To see how a test with PhenoTyper is carried out in EthoVision XT, see the sample experiment PhenoTyper hardware XT175 on the downloads section of the Noldus website (my.noldus.com). In EthoVision XT, choose File > Restore Backup and select this file. See also the document Description of sample experiments of EthoVision XT.pdf for more information.

Fear Conditioning Test

The fear conditioning test uses a classical conditioning paradigm; an initially neutral stimulus (for example, a sound or light cue) is paired with an aversive stimulus (for example, a shock or loud noise). Eventually, it is investigated to what degree the neutral stimulus can elicit a state of fear. In the fear conditioning test, you can use EthoVision XT to automatically detect freezing behavior as a measure of conditioned fear.

References

• Liu, W., Wang, X., Zhang, R., and Zhou, Y. (2009) Effects of postnatal exposure to methylmercury on spatial learning and memory and brain NMDA receptor mRNA expression in rats. Toxicology Letters, 188, 230-235.
• Pham, J., Cabrera, S.M., Sanchis-Segura, C., and Wood, M.A. (2009) Automated scoring of fear-related behavior using EthoVision software. Journal of Neuroscience Methods, 178, 323-326.

Physical Setup

The following suggestions are meant to optimize tracking:

• The camera should have a good view of the entire region the animals can be in. For zones like the shelter where the animal is not visible, you can define hidden zones.
• The lighting should be as even as possible throughout the arena and should be diffuse, so as not to cast strong shadows, which might be tracked instead of the animal.
• The background should contrast with the animal. If necessary, use a different setup for light and dark colored animals.
• Either make sure the sides of the box are not reflective (for example, by sanding), or exclude the sides from the arena. If your objects are close to the sides then you will need to track the animal when it is against the side, so the camera sees the side instead of the base behind the animal.
• If you want to use the Deep Learning technique to track the subject's nose, keep in mind that there are additional requirements. Among other things, you need a powerful graphics card. See Deep Learning: Requirements and Limitations in the EthoVision XT Help.

For More Information

• About PhenoTyper: see the PhenoTyper - EthoVision XT 17 - Reference Manual.
• About using hidden zones: see the section Shelters and other hidden zones in the EthoVision XT Help.

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. In the following sections we assume that you created an experiment using the PhenoTyper arena template.

Experiment Settings

1. Make sure that the PhenoTyper cages are connected to the EthoVision XT PC. See the PhenoTyper - EthoVision XT 17 - Reference Manual for how to connect PhenoTyper to EthoVision XT.

   Connect a third-party hardware device, which can be controlled by TTL, to one of the TTL control ports on the USB-IO Box.

2. Open the experiment and choose Setup > Experiment Settings.

3. Check that the correct cameras are selected under Video source. The procedure depends on the type and number of cameras.

   • One PhenoTyper: Select this camera and specify a saving source if you track live and also want to record a video.
   • Four or sixteen PhenoTypers with a quad unit: Select the output of the quad unit and specify a saving source if you track live and also want to record a video. Do not use a mixed image of sixteen PhenoTypers if you want to carry out nose-point, center-point, and tail-base tracking. For how to set up connections, see Camera installation in the EthoVision XT Help.
   • Four PhenoTypers without a quad unit: Select the number of cameras and select the cameras from the lists under Source. Select whether the resolution of the mixed image should be the same as the individual images, or the sum of that from the mixed images. If you also want to record video, specify that at a later stage in the Acquisition settings. Using the images from four PhenoTypers is only possible if you have the Picolo U8 H.264 board or the Picolo Alert board that receive the video output from the PhenoTypers.
4. If you want to use the Deep Learning technique to track the subject's nose, under Body point detection technique choose Deep learning. Note that in order to use this technique there are additional requirements. Among other things, you need a powerful graphics card. See Deep Learning: Requirements and Limitations in the EthoVision XT Help.
5. Under Analysis options, select Activity analysis if you are interested in freezing behavior.
6. Next to Hardware, select Use of Trial Control Hardware and click Settings.
7. Select Noldus USB-IO box in the window that opens and click OK.

8. In the Port Device Mapping window, locate the row 'SDI Port 13' and check that under Device Type, Top Unit Interface PTTI-0010 is selected. Top Unit (Standard) should be selected for each PhenoTyper that you connected to the Top Unit Interface.

   If you have connected a third-party hardware device to, for example, TTL control port 1 on the USB-IO Box, in the Port Device Mapping window, locate row 'TTL Port 1', under Device Type, select Custom Hardware and enter a Device ID (for example, 'buzzer') for this port.

9. Click OK.

For more information about setting up an experiment, see Set Up an Experiment in the EthoVision XT Help.

Arena Settings

Choose Setup > Arena Settings > open Arena Settings 1.

Click the Arena - hardware mapping button at the bottom of the Arena Settings pane.

If you only have one arena, the hardware will be assigned to this arena automatically. If that is the case, proceed directly to Trial Control Settings.

In the Arena - Hardware Mapping window, click Add Device for each device you want to add:

• For PhenoTyper: Under Device Type, select Top Unit (Standard). Enter Device Name PhenoTyper and the corresponding Top Unit (Standard) number for each arena.
• For a third-party hardware device: Under Device Type, select Custom Hardware. Enter a Device name and select a device from the list for each arena.

Click Validate Setup to validate the Arena Settings.

Trial Control Settings

With the Trial and Hardware Control add-on module in EthoVision XT, you can control PhenoTyper and third-party devices in a variety of ways. For example:

• Start recording when the animal is detected on top of the shelter.
• Stop recording when the animal has performed a specific task 50 times.
• When the animal sits on the shelter, drop a food pellet. Repeat this task 25 times.

The template experiment contains two trial control profiles:

• Default: The default Trial Control Start-Stop rule.
• Max. Trial duration 24 hrs: In this profile, a trial stops after 24 hours.

Maximum trial duration (tested):

• Without video recording: 72 hours.
• With simultaneous video recording: 3 hours.

If you need to record video of the same subject for more than three hours, we recommend that you make a series of trials.

Below you will find a few examples of Trial Control Settings. Modify the Trial Control Settings to suit your own application.

Procedure Overview

1. The trial starts with a 30-second delay (see Start trial rule and Condition 'Start delay' below).
2. Next, as soon as the mouse enters the LightSpot zone with both the nose-point and center-point, the spotlight of the PhenoTyper is turned on. When both the nose-point and center-point are not in the LightSpot zone, the spotlight is turned off. This sequence is repeated three times (see Sub-rule 'Activation LightSpot' below).
3. After the Sub-rule is completed, the trial is stopped after a 5-second delay.

Choose Setup > Trial Control Settings > New and give this a new name.

Start Trial Rule

1. To start recording as soon as the Start trial button is clicked:

   • Click the (default) Condition In zone box and press Delete.
   • Keep this Condition if you want to start recording some time after starting the trial.

Condition 'Start Delay'

1. To insert a 30-second delay after recording has started:

   1. In the Components pane, under Conditions, double-click the Time Condition or click the button next to it.
   2. In the Time condition window, type 'Delay of 30 seconds' in the Condition name field. In the Condition is met group, select After a time of 30 secs. Optionally, enter a comment and click OK.
   3. Insert the Condition box into the main sequence.

For more information about Conditions, see the EthoVision XT 17 - Trial and Hardware Control - Reference Manual.

Sub-rule 'Activation LightSpot'

3. Because the sequence of turning on/off the spotlight depending on whether the animal is inside or outside the Spot Light Zone is repeated three times, you need to create a Sub-rule for this:

   1. In the Components pane, under Structures, double-click Sub-rule or click the button next to it.
   2. In the Sub-rule window, type 'Anxiety Test' in the Sub-rule name field. Optionally, enter a comment and click OK.

4. 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.

5. 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.

6. 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.

7. 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 it to 3.
   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, configure the time condition with a delay of 5 seconds before the trial ends.

Source: EthoVision XT 17.5 Application Manual - Other Conditioning Tests