Knowledge Base

Analyzing Intervals in the Fear Conditioning Test

Define the Intervals with the Nesting Function

By default, the whole trial timeline is selected for analysis. However, you may want to select part of the timeline, for example to analyze the Activity variable only when the white light was on. To do so, in the Data profile define an interval based on the event "White Light On".

1. Choose Analysis > Data Profile > New.
2. Under Nesting, click the option corresponding to the type of interval you want.
3. Specify the interval and place the Nest box between the Start and Results box.

Which options shall I choose?

To analyze the behavior of the subject when, for example, the white light was on, choose Trial Control State. In the window that appears, select the action that marks the start of the interval (e.g., Light On - becomes active), and the action that marks the end of the interval (Light Off - becomes active).

If you want to analyze a complex interval, from A to B, where A and B are events of different type, for example, behavior and activation of a stimulus, choose Free interval.

For example, select the time from the end of the shock (Shock Off - becomes active) and the first instance of freezing (detected using Activity or Activity state).

You can also combine events with time. For example, select the 30 seconds interval immediately after the end of the Shock. From Trial Control - Action: Shock Off - becomes active to Time - Elapsed time: 30 s after start event.

Visualize the Intervals

Once you have defined the intervals for analysis, it is always a good idea to plot the data and check that the intervals defined in the previous steps are correct.

1. Choose Analysis > Results > Plot Integrated Data.
2. The data selected within the intervals is shown in full colors while the data outside the intervals is shown in dimmed colors. Check that the intervals defined correspond to the time segments that you want to analyze.

In the example, Activity is visualized within an interval defined as "from Tone On to Shock On".

If the intervals are defined correctly, proceed with the statistics calculation.

Calculate the Statistics

Choose Analysis > Results > Calculate Statistics and click the Calculate button. The results table shows the statistics of the dependent variables defined in the Analysis profile, within the intervals defined in the Data profile.

For More Information

• For details about the analysis variables, see Dependent Variables in Detail in the EthoVision XT Help (F1).
• For more examples about analyzing intervals, see Analyze Track Segments in the EthoVision XT Help (F1).
• For more examples of control of hardware devices and PhenoTyper, see the chapter Conditioning Tests in this manual.

Technical Specifications

Infrared Top Light and Controller

For the technical specifications of the Fear Conditioning system, see the Instruction Manual FCS 46000.pdf that comes with that system.

Noldus Device Numbers

• Top light: UBIRT-001x
• Controller: UBIRC-001x

Voltage: 24V (standard Noldus power supply)

Current consumption on max LED intensity: 220mA

Light wavelength: 940nm

Incorporated camera: 1280 x 1024 pixels at max 60 fps (30 fps is recommended)

Dimensions (L x W x H):

• Top light: 28.6 x 29.3 x 8.5 cm (11 17/64" x 11 17/32" x 3 11/32")
• Controller: 8.0 x 4.0 x 4.2 cm (3 5/32" x 1 37/64" x 1 21/32")

Weight:

• Top light: 860 g (30.3 oz)
• Controller: 50 g (1.76 oz)

Power adapter: Type number TRH70A240-01E13VI, Input 100-240V 1.5 A, Output 24V 3.0 A

Other Conditioning Tests

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 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 https://noldus.com/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 (www.noldus.com).

To see how a test with PhenoTyper is carried out in EthoVision XT, see the sample experiment PhenoTyper hardware XT180 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

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 (e.g., 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 19 - Reference Manual.
• About using hidden zones: see the section Shelters and other hidden zones in the EthoVision XT Help.

A Conditioning Test in EthoVision XT

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 19 - 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. Select whether the resolution of the first camera and that of the mixed image. Note that depending on the number of cameras and the frame rate, you may need to reduce the resolution of the mixed image. For more information about installing and connecting multiple cameras, see Camera installation in the EthoVision XT Help and the PhenoTyper - EthoVision XT 19 - Reference Manual.
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 want to measure freezing behavior.
6. Next to Hardware, select Use of Trial Control Hardware and click Settings.
7. Select Noldus USB-IO box.
8. In this next step, you define the ports of the USB-IO box connected to PhenoTypers. Select SDI port 13, then Top Unit Interface. Select Top Unit (Standard) for all the rows below (depending on how many PhenoTypers are connected to a Top Unit Interface). For PhenoTyper 2, and for more details and examples, see the PhenoTyper - EthoVision XT 19 - Reference Manual. If you use third-party devices, also select the TTL ports that are connected to those devices.
9. Click OK.

For More Information

• About port mapping: see the EthoVision XT 19 - Trial and Hardware Control - Reference Manual.
• About PhenoTyper connections and devices: see the PhenoTyper - EthoVision XT 19 - Reference Manual and the PhenoTyper - EthoVision XT 19 - Service Manual for specific devices like the Pellet dispenser and the Lickometer.
• About general experiment settings: 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 with Trial Control Settings below.

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) # 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 4. 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 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- and center-point, the spotlight of the PhenoTyper is turned on. When both the nose- 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'

2. 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 19 - 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/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 the relevant body points.

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Source: EthoVision XT 19 - Application Manual