Knowledge Base

Introduction

Principle of Operation

Fear Conditioning

In a typical Fear Conditioning paradigm, single-frequency sounds are delivered as a neutral or conditioned stimulus (CS). Experimental procedures where the CS is administered are usually named Cued Fear Conditioning. Electric current from the grid floor is delivered as the naturally aversive, or unconditioned stimulus (US).

This chapter describes how you can use EthoVision XT 17.5 and later with the Ugo Basile Fear Conditioning System NG-series 46000, to carry out fear conditioning experiments.

If you use a previous version of EthoVision XT, please refer to the EthoVision XT Application Manual for that version.

For an overview of Fear Conditioning, and how fear is measured in EthoVision XT, see the white paper which you can download from our web site: www.noldus.com/animal-behavior-research/fear-conditioning

Controlling the FCS Stimuli from EthoVision XT

1. Connect the EthoVision XT computer to the Ugo Basile Fear Conditioning System (FCS). For details, see Physical Setup below.
2. In EthoVision XT, create your experiment and declare the devices that you are going to use. Next, program the activation of the devices in the Trial Control Settings. For details, see The Fear Conditioning Experiment in EthoVision XT below.

   If you have multiple FCS cubicles, you can control the stimuli in each cubicle independently.

3. Carry out data acquisition. See Acquire the Data below.

Experiment Devices and Parameters

You can specify the devices and their parameters directly in EthoVision XT. This also makes it easier to test a device.

• Shock, with adjustable current intensity.
• Tone, with adjustable frequency and volume.
• White noise, with adjustable volume.
• Visible (white) light, with adjustable light intensity.
• IR light, with adjustable light intensity.
• Fan, which can be switched on and off.

You can also have EthoVision XT check if the door of the FCS cubicle is open or closed.

Note: If you work with EthoVision XT 16 or earlier, you must connect the EthoVision XT computer to the Ugo Basile Touchscreen Controller 40500-001. Please refer to the EthoVision XT Application Manual for that version.

Analysis of Freezing

The detection of freezing can be automated and based on analysis of the variable Activity in EthoVision XT. For details, see Activity and Activity State in the EthoVision XT Help, under Dependent Variables in detail.

Changes Relative to Previous EthoVision XT Versions

With EthoVision XT 17.5 and later versions you can control the Ugo Basile FCS without connecting the touchscreen controller. For new systems, the cabling is largely reduced and the system is easier to troubleshoot.

• Cable connections are simpler - from the PC directly to the FCS cubicles through USB.
• When you create an EthoVision XT experiment, the devices of the Ugo Basile FCS are automatically added to the experiment.
• In the Trial Control Settings, you can easily recognize the names of the devices (e.g. Tone) instead of for example Line 1, as it occurred in the previous EthoVision XT versions.
• You can test the devices directly in the Trial Control Settings, before carrying out the actual tests.

If You Have the "Old" FCS Setup

It may be that you have the FCS setup connected to EthoVision XT through the Noldus USB-IO box, and that you have upgraded to EthoVision XT 17.5. In this case you can keep using the "old" connections. For the instructions, see the EthoVision XT 17.0 Application Manual, which you can find on the downloads page of my.noldus.com.

Alternatively, contact Noldus to purchase the hardware necessary for the current setup.

For General Information

EthoVision XT Help

For more details, choose Help > Help Topics or press F1 in the software.

Other Documentation

On the Windows Apps screen, choose Noldus > EthoVision XT 17 Other Documentation. See in particular:

• For information on programming trial control, conditioning routines, actions and conditions, and the USB-IO box: the EthoVision XT 17 - Trial and Hardware Control - Reference Manual.
• For information on conditioning tests and examples: see other chapters in this manual.

You can find these and other manuals on the Noldus website at my.noldus.com. Note that you need to log in first to access the downloads page.

About the Ugo Basile Fear Conditioning System

This system comes with the instruction manual Fear Conditioning Systems-NG Series 46000, in PDF format.

Technical Support

If you encounter problems, browse to my.noldus.com, and contact the help desk.

Physical Setup

One-Cubicle Configuration

Note: The diagrams only include the connections essential for communication between EthoVision XT and the FCS. See also the Ugo Basile Fear Conditioning Systems NG Series instruction manual.

Step-by-Step Instructions

1. Connect the EthoVision XT computer to the Ugo Basile FCS using the appropriate USB cable. You need a USB cable with a type-A connector at one end and a type-B connector at the other end.
2. Connect the camera to the PC.

   If your computer has an Ethernet board with one or two ports, you need a Power over Ethernet (PoE) injector.

   • Connect one of the ports of the Ethernet board of the computer to the PC port (IN) on the PoE injector.
   • Connect the camera port on the back of the cubicle to the CAM port (OUT) of the PoE injector.
   • Next, connect the PoE injector to a power outlet.

   If your computer has a 4-port Ethernet board installed, and if this board receives power from the PC internal power supply, you do not need a PoE injector. Connect the camera directly to one of the board's Ethernet ports. See the EthoVision XT Help for details.

3. The cubicle is equipped with an IR light box above the conditioning cage, and an IR light controller to adjust the IR light intensity. Connect the red and black cable that comes from the cubicle to the IR light controller.

   Connect the IR light controller to a power outlet through its power adaptor. Attach the IR light controller as indicated on the labels.

   If you work with multiple cubicles, connect the IR controller as explained above for all the cubicles.

The Video Camera

In most cases the camera is embedded in the IR light box at the top of the cubicle.

If you have not done so yet, you must configure the IP address of the camera and of the Ethernet port on your PC. You can find the IP address of the camera on the front panel of the cubicle's light box.

For details on how to install and configure the EthoVision XT cameras, see Camera Installation in the EthoVision XT Help.

To adjust the camera orientation, see Camera Settings below.

Four-Cubicle Configuration

Step-by-Step Instructions

1. Connect each cubicle to the EthoVision XT computer through the Ugo Basile USB Hub.

   For this you need 5x USB cables with a Type-A connector at one end and a Type-B connector at the other end.

2. Connect the Ugo Basile USB Hub to the power outlet using the appropriate power adaptor.
3. 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.
4. 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.

5. Connect one IR controller to each cubicle, as described in the one-cubicle procedure above.

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).

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.

If you have the Ugo Basile FCS equipped with USB cameras, connect the cameras directly to the EthoVision XT computer.

For details:

• 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 lens, F2.0, 1/1.8".

How to View the Live Camera Image

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

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.

If the image is slightly out of focus, contact Noldus Support for how to adjust the focus of the camera lens.

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. See below for the values recommended.

Remember to change the settings for all the cameras.

Image Brightness

If the image from the camera is too dark, turn the knob of the IR light controller all the way to the right. In most cases this is enough to make detection optimal. If that is not the case, you can increase the camera Exposure time, or the Gain.

The Ugo Basile FCS also has an IR light 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.

• Analog gain: 1
• Color space: Mono8 (Y800)
• Resolution: 1280 x 1024
• Acquisition Frame rate: 25 or 30
• Exposure: 16000 s

EthoVision XT Settings

Prerequisites

Make sure that all devices are connected and powered up.

Create an EthoVision XT Experiment

1. Start EthoVision XT.
2. Choose File > New From Template, then click Apply a pre-defined template.
3. On the How is the arena configured page, select Open field square, with No zone template. Next to Number of arenas, choose 1 for a 1-cubicle configuration, 4 for a 4-cubicle configuration.
4. When you are asked How many subjects per arena will you track, select 1 subject.
5. Complete the 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.
3. Next to Merged camera view, select 1280 x 1024 or similar.

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. In most cases 1, 2 or 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.
2. Click the Video Tutorial button next to that option for an overview of Activity analysis.

Specify the Ugo Basile Fear Conditioning System

Important: Make sure that the Number of Arenas selected corresponds to the number of FCS cages used in that experiment before following 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 (see COM Port Communication below for how to find out which COM port is associated to a specific USB physical connection).
4. Important: Take note of which cubicle is connected to which COM port. Make sure that only the necessary COM ports are added. If you added accidentally a wrong COM port, select that and click Delete.
5. When finished, click OK and proceed with arena settings.

COM Port Communication

In order to know which COM port is associated with a specific cubicle, do the following:

1. In Windows, open the Device Manager.
2. Open the item named Ports (COM and LPT).

   If the FCSs are connected physically to the PC through the Ugo Basile USB Hub and powered up, you should see one up to four items USB Serial Port (COMx): COM11 to COM14.

   Tip: If you do not see the Ports item, from the View menu of the Device Manager select Devices by type.

3. Disconnect the USB cable from one of the FCS cubicles.
4. Look at the Ports (COM and LPT) list. One of the items has disappeared. That is the COM port currently associated with that cubicle.

Source: EthoVision XT 17.5 Application Manual - The Fear Conditioning Test