Knowledge Base

The Fear Conditioning Test

Hardware Setup and Camera Connection

Connecting Cameras and Light Units

1. Connect each camera to one of the ports of the Ethernet Interface board in the EthoVision XT computer. For this you need 4x Ethernet cables with RJ45 connectors.
2. Connect the Top Light Units to the power outlet through the appropriate power adaptors.

   For more information on how to install and configure the Ethernet Interface board, see Install the GigE cameras in the EthoVision XT Help.

3. Connect one IR controller to each cubicle.

   Note: The external IR light controller is no longer necessary if you updated the cubicle. See update the cubicle firmware section below.

Connect Multiple Cameras

If your EthoVision computer is provided with a 4-port Ethernet interface board for connecting multiple GigE cameras, the cameras can get the power from the PC itself. If you purchased the computer and Ethernet board from Noldus, the board is already powered by the PC. Otherwise, open the computer casing and connect the Ethernet interface board with the internal power supply of the computer.

Once power is provided through the PC, connect each camera to one of the ports of the Ethernet interface board. Label the ports and the cables so you always know which port is connected to which camera (and which cubicle).

IP Addresses

Note: If you have the Ugo Basile FCS equipped with USB cameras, connect the cameras directly to the EthoVision XT computer. Please skip this section.

If your FCS is equipped with Basler GigE cameras through Ethernet connections, each camera must have an IP address that matches the IP address of the Ethernet interface board on your PC.

The basic rules are:

• The first two numbers are the same for all cameras: 192.168.
• The third number should be the same and unique for each camera-Ethernet port combination.
• The fourth number can be any value, but must differ between camera and Ethernet interface board, and should suggest which cubicle (arena) it refers to.

For example:

• 192.168.1.101 for camera 1 (cubicle 1) and 192.168.1.51 for Ethernet port 1 of the Ethernet interface board.
• 192.168.2.102 for camera 2 (cubicle 2) and 192.168.2.52 for Ethernet port 2 of the Ethernet interface board.

The IP address of each camera can be found on the front panel of the cubicle's light box. You need those addresses to configure the cameras.

For details about assigning IP addresses:

• In the HTML help: choose Help > EthoVision XT Help > Camera installation > Install GigE cameras.
• In the Video Tutorial: choose Help > Video Tutorial > Set Up the Cameras.

Camera Settings

Camera Type and Lens

The camera used in Noldus systems for Fear Conditioning is a Basler camera acA1300-60gm with a 2.95 mm fixed focal lens, F2.0, 1/1.8".

How to View the Live Camera Image

In order to view the camera image, you must activate the IR light in the cubicle. The camera has an IR-pass filter that blocks visible light.

1. Make sure that the camera has been configured and connected to the PC.
2. Start EthoVision XT and create a new experiment (File > New) or open an existing experiment without trials acquired.
3. Choose Setup > Experiment Settings.
4. Under Video Source, select Live tracking.
5. Select the camera from the list under Source.
6. Click the camera button for the live view.

Camera Orientation

The camera has been optimized at Noldus before shipping, therefore in most cases you do not need to adjust its orientation. If, for any reason, the video image is not optimized, follow the instructions below.

Camera Alignment

This section applies for previous FCS versions which had Noldus IR light box on top.

Turn the three screws indicated to alter the camera orientation. If you tighten one of the screws, the camera points more to the other side. Loosen the same screw to have the opposite effect. For example, tighten screw b to have the camera pointing more to the back of the cubicle. Check the resulting image in EthoVision XT; make sure that the bottom of the cage is aligned at the center of the camera view.

Camera Software Settings

The camera frame rate, exposure time and other settings can be adjusted in EthoVision XT, or in the camera software Pylon Viewer.

In EthoVision XT, choose Setup > Experiment Settings. Click the camera button and locate the settings you want to change.

For multiple cameras, you need to adjust the settings for the first camera. Then, adjust the resolution of the Merged view.

Video Resolution and Camera Zoom Level

The camera lens of the FCS is a fixed focal lens. To adjust the zoom level, adjust the video resolution (in pixels) so that a portion of the maximal camera view is recorded. Note how the resolution setting crops the image.

• Analog gain: 1
• Color space: Mono8 (Y800)
• Video resolution single camera (H x V): 640 x 480
• Video resolution merged view (H x V): 1280 x 960
• Acquisition Frame rate: 25 or 30
• Exposure: 16000 us

In a typical example, EthoVision XT makes a video of 1600 x 1200 from four cameras set to 800 x 600.

Important: Whenever you change the resolution, open the Arena Settings and update the background image for your arenas before acquiring the data.

The Binning Option

If you need to reduce the video resolution without cropping the image, you can use the Binning option in the Video Settings of EthoVision XT.

In EthoVision XT, choose Setup > Experiment Settings. Click the camera button and in the Video Settings window click the General tab. Select the Binning option and leave the value at 2. The Binning option reduces the number of pixels of the single camera image by half but does not crop the image.

If you choose to select Binning, note that the brightness of the image increases. Make sure that this does not have negative effects on detection. If the image is too bright, reduce the Exposure time or the Gain.

The Binning option must be the same across cameras. Remember to select (or de-select) the Binning option for all cameras, before setting the resolution of the merged view.

Image Brightness

If the image from the camera is too dark, do one of the following based on what FCS version you have:

• If you have the IR light controller attached to the cubicle, turn the knob of the IR light controller all the way to the right. In most cases this is enough to make detection optimal.
• Increase the IR light intensity up to 100% with the Trial Control Settings in EthoVision XT.
• Increase the IR light intensity using the software on the tablet PC.
• Place white plates at the sides and at the bottom of the FCS cage to increase contrast with dark-fur animals. Place dark plates in case of white animals.

To increase the brightness of the image further, you can adjust camera settings in EthoVision XT or Pylon Viewer:

• Increase the camera Exposure time. Be aware that a too high Exposure time may conflict with the camera frame rate. EthoVision XT shows a message in the Video Settings window if that is the case.
• Increase the Gain to 1 or 2. Note that a higher Gain also creates more image noise.
• Another option to increase the brightness of the image without increasing noise is to set the image Binning Horizontal and Binning Vertical to 2. Make sure that the Binning Mode is set to Summing for both. However, with binning the video resolution (that is, the width and the height of the camera image, in pixels) is half of the original.

Older versions of the Ugo Basile FCS have the IR light source placed at one of the top corners of the cubicle. You do not need to operate this light if you have the IR light box above the conditioning cage.

Upgrade Your FCS System

Aim

To adapt the Fear Conditioning System to use in combination with EthoVision XT 17.5 and later.

• The touchscreen controller is no longer needed.
• You can adjust the FCS settings (e.g. tone frequency, shock current) directly in EthoVision XT.
• The time needed for installation and setting adjustment is about one hour.

Prerequisites

• You have received the following from Ugo Basile:
  • 1x upgrade box for each cubicle.
  • 1x power cable, 1x USB cable, 4x color-coded cables for each cubicle.
  • Two hexagonal Allen keys.
• Ugo Basile needs the touch screen controller back first, so that they can copy the settings of the touch screen controller / Fear Conditioning box to the upgrade box. That way you can work with the same settings as before.

Procedure

1. Remove the four screws at the corners of the upgrade box and remove the cover plate.
2. Locate the holes on the back panel of the cubicle.
3. Attach the box to the back panel. Make sure that the cable connections are on top.
4. Put the lid of the box back in place and tighten the screws.
5. Connect the color-coded cables as shown in the installation diagram.
6. Finally, connect:
   • The upgrade box to the PC (or USB hub, in case of 2 or more cubicles) using the USB cable that came with the upgrade box.
   • The upgrade box to a power outlet.
   • The camera to the PC's Ethernet board.
7. Repeat the steps above for the remaining cubicles.
8. Next, follow the steps in The Fear Conditioning Experiment in EthoVision XT section below.

Update the Cubicle Firmware

Aim

To make the FCS cubicles compatible with the new IR illumination system that is fully controllable from EthoVision XT.

Important: Follow this procedure only when requested by Noldus Support.

What You Need

• The firmware updater installation files: FW_Updater Setup.msi and setup.exe.
• The new firmware file Cubicle.elf_7.34.S19.

Procedure

1. On the EthoVision XT full installation package, open Drivers and Tools > Utilities > Ugo Basile FCS.

   You can download the full installation package from my.noldus.com.

2. Copy the file Ugo Basile FCS FirmWare Updater [version number].zip to your EthoVision XT PC and extract its contents.
3. Double-click the file setup.exe. Complete the installation procedure.
4. Power up the cubicle and connect it to the EthoVision XT PC through the USB cable.
5. Start the application.
6. Choose Select Firmware.
7. In the window that opens, choose the file Cubicle.elf_7.34.S19.
8. Click Firmware Update.
9. If the current version is 2.27 or newer, the update process should start automatically. Otherwise, you need to:
   • Disconnect both the power supply and the USB cable from the cubicle.
   • Reconnect both the power supply and the USB cable, approximately at the same time.
10. Wait until the Erase and the Programming processes are completed.
11. After the device has rebooted, you can change the tab to BOARD SETTINGS.
12. Leave or set the Device ID to 1 and set the Default IR intensity to the desired value (recommended: 100%). This will be the default intensity at startup.
13. Click Save to Board.

Check the Ugo Basile Interface for EthoVision XT

1. Open the Control Panel > Programs and Features.
2. Check that the software Noldus - HardwareInterface UgoBasile - x64 package version 1.0.14 or later is installed.

   If a previous version is installed, uninstall it. Next, copy the file HardwareInterfaceUgoBasile - X64 Package - [version number].msi from the EthoVision XT 18 full installation package, under Drivers and Tools > Prerequisites > HardwareInterfaceUgoBasile to your PC, then run this file.

Test the IR Light

To test the IR light, in the Trial Control Settings create an Action based on the IR Light. Set the intensity to 100% and click Test. Click OK then check in the Detection Settings whether the IR light is still on.

The Fear Conditioning Experiment in EthoVision XT

Prerequisites

Make sure that all devices are connected and powered up.

Create an EthoVision XT Experiment

1. Start EthoVision XT and choose File > New From Template, then click Apply a pre-defined template.
2. On the How is the arena configured page, select:
   • If you work with one or two cubicles, select Fear Conditioning System 1 cubicle. Next to Number of arenas, select 1 or 2.
   • If you work with four cubicles, select Fear Conditioning System 4 cubicles. Next to Number of arenas, select 4.

   Note: This template does not include pre-defined zones as they are not used often in Fear Conditioning.

3. When you are asked How many subjects per arena will you track, select 1 subject.
4. Complete the guided procedure and give the experiment a name. Next, click OK.

Experiment Settings

Cameras

1. Choose Setup > Experiment Settings.
2. Under Video Source, next to Number of sources, select how many cameras (one per cubicle) you are working with. In the list under Source, select the name of each camera.

   The IP address should indicate which camera belongs to which cubicle, provided that you have assigned IP addresses correctly.

3. Next to Merged camera view, select 800 x 600 (cropped view of the cage) or 1280 x 1024 (full view of the cage).

Number of Arenas and Tracked Features

Important: Do this before selecting the Ugo Basile FCS in the Trial Control Hardware Settings.

1. In the Experiment Settings, under Arenas, select the Number of Arenas you use, that is, the number of cubicles (1 to 4).
2. Under Tracked Features, in most cases Center-point detection will suffice for fear conditioning experiments.

Activity Analysis for Freezing Detection

1. In the Experiment Settings, under Analysis Options, select Activity analysis if you want to analyze freezing behavior.

   Tip: Click the Video Tutorial button next to that option for an overview of Activity analysis.

Specify the Ugo Basile Fear Conditioning System

Important: Have you already selected the number of arenas? Make sure that the Number of Arenas selected corresponds to the number of FCS cages used in that experiment. Only after that, follow the instructions below.

1. In the Experiment Settings, under Trial Control Hardware, select Use of Trial Control Hardware, and click Settings.
2. In the Hardware Interface window, select Ugo Basile F.C. Controller.
3. In the Fear Conditioning System COM Port Settings window, click the Add button.
   • Select a COM port and click OK. Repeat this step to add the COM ports connected to all the FCS cubicles.
   • For a setup with the Ugo Basile USB Hub and four cubicles, add COM11, then COM12, COM13, and COM14.
   • For a setup with one cubicle connected directly to the PC, select the COM port associated with that cubicle. If you have created the experiment from a template, there may be one or more COM ports already selected in the Fear Conditioning System COM Port Settings window. If they do not match those associated with the cubicles of your setup, delete them and add the correct ones.
4. When finished, click OK and proceed with arena settings.

Important: Take note of which cubicle is connected to which COM port. Make sure that only the necessary COM ports are added. If you accidentally added a wrong COM port, select that port and click Delete.

Source: EthoVision XT 19 - Application Manual