Knowledge Base

The Fear Conditioning Test

The Fear Conditioning experiment in EthoVision XT 193

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:

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 on page

182. 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 The Fear

Conditioning experiment in EthoVision XT on page 193. If you have multiple FCS cubicles, you can control the stimuli in each cubicle independently. 3. Carry out data acquisition. See Acquire the data on page 212. The Fear Conditioning Test

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 way simpler - from the PC directly to the FCS cubicles through USB. The Fear Conditioning Test 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. See Physical setup on page 182.

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. The Fear Conditioning Test 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. The Fear Conditioning Test

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 (see also the figure on the previous page).

• 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 (see the figure below) to the CAM port (OUT) of the PoE injector.
• Next, connect the PoE injector to a power outlet. The Fear Conditioning Test 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 a 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. The Fear Conditioning Test 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 yet so, 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 on page 190. The Fear Conditioning Test

four-cubicle configuration

1 - Cubicles

2 and 3 - Video cameras and IR light controllers

Step by step instructions

See the schemes on the previous pages. 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 (see the top figure above). The Fear Conditioning Test 3. Connect each camera to one of the ports of the Ethernet Interface board in the EthoVision XT computer (see the figure below). For this

you need 4x Ethernet cables with RJ45 connectors. See also 2 in the

figure on page 187. 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 procedure on page 184. See also 3 in the figure on page 187.

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. The Fear Conditioning Test Once power is provided through the PC, connect each camera to one of the ports of 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". The Fear Conditioning Test

How to view the live camera image

1. Make sure that the camera has been configured and connected to the PC (see above).
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. The Fear Conditioning Test 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 the 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 (see page 184). 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 a 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

Color space

Mono8 (Y800)

Resolution

1280 x 1024

Acquisition Frame rate

25 or 30

Exposure

16000 s The Fear Conditioning Test

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.
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. The Fear Conditioning Test

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 Have you already selected the number of arenas (see

above)? Make sure that Number of Arenas selected corresponds to the

The Fear Conditioning Test 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 (see com port communication on page

196 for how to find out which COM port is associated to a specific USB physical connection).

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. 4. When finished, click OK and proceed with arena settings: define the arenas on page 197. The Fear Conditioning Test

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 (see page 186) 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. For example, after disconnecting cubicle 4 (COM14):

tip Number the FCS cages and cubicles, and take note which COM port

is linked with each cubicle. That makes troubleshooting easier. The Fear Conditioning Test If, after disconnecting a cubicle, a COM port other than the expected one disappears from the list, it may be that cables are swapped between the ports in the USB Hub and the corresponding cubicles.

arena settings: define the arenas

Create and calibrate the arenas

1. Choose Setup > Arena Settings.
2. Grab a background image from the cameras, calibrate and draw the arenas. For details, see Arena Settings in the EthoVision XT Help.

tip To quickly calibrate, draw lines between opposite walls of the

cage, next to the grid, and enter 17 cm (the cage width). See page 192 for tips how to get a bright image of the cage. 3. If the image of a cage is not centered, see Camera orientation (page 191). 4. Proceed with declaring the FCS devices for each arena (next section). The Fear Conditioning Test

arena settings: declare the fcs devices

Follow this procedure to specify the FCS devices that you want to use for each cage (arena). 1. In the Arena Settings, click the Arena - Hardware Mapping button at the bottom-right corner of the EthoVision XT screen. 2. The Arena - Device Mapping window opens.

This window lists under Device groups the FC systems connected to

the COM ports currently declared (page 194). The second column, Arena, lists the arenas currently associated with each system. 3. Check carefully that the name of each arena matches the physical FC system (and the COM port associated with it). If the two do not match, select the correct arena name under Arena. 4. Once you are ready, click OK. The Arena-Hardware Mapping window opens. The Fear Conditioning Test This window lists the names of the actual devices that EthoVision XT can control. By default, each device is uniquely associated automatically to one arena based on the scheme in the window that opened in the previous step.

For example, all the devices of Fear Conditioning System 1 at COM 1

are listed under Arena 1. - The column Device type shows the types of device currently selected. If the type you want to use does not appear, add a new device (see page 200). - In the column Device name you find names like Device A, Device B, etc. These are generic names for a specific type of device, which you find back when you program the activation of the devices in the Trial Control Settings. If you like, you can change them to Shock, Tone, etc. - In the columns Arena 1, Arena 2, etc. you find the list with the names of unique physical devices. These are automatically assigned based on which FCS system connected to a specific COM port is associated with which arena in EthoVision XT (see through the COM ports (see above). Your task now is to assign each physical device to a specific arena. 5. important Test the devices! See page 203. You can also test the devices in the Trial Control Settings.

note The item UB FCS IR Light listed in the Arena - Hardware Mapping

window is for controlling the IR lamp located at the top-left corner of the cubicle, not the IR light given by the light box. You can control the The Fear Conditioning Test IR light of the light box by turning the knob of the IR controller (page 187).

Add a new device

If the FCS devices are not listed in the Arena-Hardware Mapping window, do the following: 1. Click the Add device button. 2. Under Device type, select a device (e.g. UB FCS Tone). 3. Under Device name, either accept the default name or enter a new, meaningful name, for example Tone. - If you plan to activate a device type in all the cages at the same time, a generic name like Shock or Tone is fine. - If you plan to activate a device type in different cages at different

times, it is a good idea to specify a unique Device name, for

example Tone for Cage 1, and assign that device only to the arena associated with that cage. 4. Locate the column for the arena you want to assign the device to, and select the physical device from the drop-down list. For example,

under Arena 1, select UB FCS Shock 1; under Arena 2, select Ugo Basile

FCS Shock 2, etc. How many devices are available in a list depends on how many COM ports you have declared (see page 195). Make sure you do not select the same unique device under two or more arenas. The Fear Conditioning Test

examples of arena-hardware mapping

EthoVision XT controlling one cage

note The item UB FCS IR Light is for controlling the IR lamp at the top-

left corner of the cubicle, not the IR light box.

EthoVision XT controlling four cages - 1

Use this configuration whenever you plan to administer a stimulus in all the FCS cages at the same time, for example, to activate the tone in all the cages exactly one minute after the start of the trial.

Define a Device name that is valid for all the cages (arenas). Under

Arena 1, Arena 2, etc., select the physical devices for each of those

arenas.

In the following example, Device A represents the foot shock for all the

cages. This means that when you use a hardware action Device A -

Shocker On in the Trial Control Settings (see page 206), that action is

carried out in all the cages. Make sure not to select the same physical device twice for two arenas. Repeat the steps for the remaining devices. The Fear Conditioning Test

note You can also use this configuration when tracking starts at

different times in different arenas, for example when you release the subject at different times and EthoVision starts tracking after the subject has been detected.

EthoVision XT controlling four cages - 2

Sometimes you need to apply different protocols to different cages (arenas). For example, to provide a stimulus after the subject has been immobile for one minute. Or, give a shock in cage 1 after 1 minute and give a shock in cage 2 after a random time, or the like. In such cases the activation of a stimulus device is cage-specific. You can create this configuration by linking a specific device name (e.g. Device A) only to a specific physical device in one of the arenas (e.g. Arena 1). Device A should not be assigned to other arenas. Therefore, you must add four new devices (see page 200) of the same type, and assign each of them to one arena. For example, in the case of UB FCS Tone: Device A: UB FCS Tone 1 under Arena 1. Device B: UB FCS Tone 2 under Arena 2. Device C: UB FCS Tone 3 under Arena 3. Device D: UB FCS Tone 4 under Arena 4. The Fear Conditioning Test Furthermore:

Remember to select in the remaining cells of

the table. Repeat the steps for the other device types that you want to use. For more information about applying different protocols to

different cages, see Applying different protocols to different arenas

in the EthoVision XT 17 - Trial and Hardware Control - Reference Manual.

test the fcs devices

Prerequisites

All the connections are set as in the section Physical setup. You have created an experiment and have specified the FCS in the Experiment Settings (see page 194). You have drawn and calibrated the arenas (see page 197). You have specified the FCS devices for each arena (see page 198).

Procedure for actions

This applies to Visible Light, Tone, Shocker, White Noise, Fan, IR Light. 1. In EthoVision XT, choose Setup > Trial Control Settings and open one of the existing profiles. The Fear Conditioning Test 2. In the Components pane, under Actions, click the button next to the name of the device you want to test. 3. In the window that appears, select the device, the type of action, adjust its settings, and click Test. 4. Verify that the devices works as expected. Next, click Reset to reset all devices to their default values. If the action is carried out in another cubicle, it means that the wrong device has been selected in step 3, or that the devices have not been mapped correctly in the Arena Settings (see page 198).

Procedure for the UB FCS door

This applies to the device UB FCS Door. 1. In EthoVision XT, choose Setup > Arena Settings and click the Arena-Hardware Mapping button. 2. In the Arena-Hardware Mapping window, select the row for UB FCS Door. Next, click the Test button. 3. The Test UB FCS Door window opens on the screen. Open/close the door of the corresponding cubicle and check the message displayed in the window (Door Open? True or false). The Fear Conditioning Test

Verify that the shocker actually gives a shock

Place a LED diode between two bars of the shocker grid, or touch a bar with a phase detector. A phase detector. If the shocker is connected correctly and working, the LED turns on. You can also touch the grid bars, however a current intensity around 1 mA is the threshold of feeling. A lower current may not be felt.

Measure the amplitude of the shock

important For this you need an oscilloscope and an oscilloscope probe

with insulated channel and a 100 K resistor. 1. Set the oscilloscope to 50V/DIV and time base 10 ms. 2. Connect the oscilloscope probe with a 100 K resistor to the shock grid, as indicated in the following picture. 3. In EthoVision XT, in the Arena - Hardware Mapping window, select

the shock device for a specific cubicle and click Test. Set the shock

intensity to 1.5 mA. The Fear Conditioning Test 4. Click the Test button to activate the shock. You should see a square wave on the display of the oscilloscope. Read the amplitude there; remember that with the current settings one vertical division corresponds to 50 V. Once you know the voltage, the current is equal to the voltage divided by the resistance (in this example 100000).

trial control settings

Follow this section to learn how to program EthoVision XT to send commands to the FCS devices at the right time during data acquisition.

Basic structure: Actions and Conditions

To activate /deactivate a stimulus, use the Actions function. In general,

two actions must be separate by some time. Here you find an example of activation of a stimulus for one second. 1. Choose Setup > Trial Control Settings. 2. In the Components pane, under Actions click the button next to the device name that you want to use, and specify the type of action. 3. In most cases you need to add a number of boxes in a sequence. For

example, the sequence Shock On (Action) > Time Condition > Shock

Off (Action) define the activation of the shock for a specific time set

in the Time Condition. 4. Click and drag from the center of one box to the next to connect the boxes with the arrows. Depending on how you map the devices on the Arenas (see page 201), this action can be carried out in one or more cubicles simultaneously. The Fear Conditioning Test

Recurring stimulation using Sub-rules

In most cases you need to administer a stimulus at regular intervals, for example every minute. If you repeat the sequence depicted in the figure above, one for each round, you would end up with a large

number of boxes to manage. Instead, create a Sub-rule that contains

the actions that you want to repeat. Specify how often and when the routine is repeated in the Sub-rule settings. In the following example, a Sub-rule with the same instructions as above will be repeated 15 times. The shock is given every minute. After

the start of the trial, the software waits one minute (box A), then the

Sub-rule reference box (B) activates the Sub-rule (C). The stimulus is

given as above (D-E-F). A time condition of 59 seconds (G) ensures that

the next stimulus is given exactly one minute later (1 s of shock + 59 s = 60 s). The Sub-rule is repeated 15 times as specified in the Settings of box B. After that, the trial stops (box H).

Analyze data using Trial Control events

If you want to analyze the data in specific time segments, you can use the actions and conditions of Trial Control Settings to define specific intervals. See analyze intervals on page 215.

For more information

For more information and examples of actions, conditions and sub- rules, see the EthoVision XT 17 - Trial and Hardware Control - Reference

Manual. To open this manual, choose Apps > Noldus > EthoVision XT 17

Other Documentation. The Fear Conditioning Test

detection settings

Release the subjects in the FCS cages. Choose Setup > Detection Settings.

Detect the subject

In the Detection Settings window, click Automated Setup. Draw a

rectangle around each subject in the video image, and follow the instructions on the screen.

Minimize the indentations caused by the grid

In the Detection Settings, click Advanced. Under Subject Contour,

choose what follows depending on the color of the subject and the grid bars: If the grid bars are the same color as the subject, use the first

Erosion filter. Increase the value near Erosion until the grid is no

longer detected. Normally 2-3 pixels are fine. Check that the whole body is detected, indicated by the yellow blob. The Fear Conditioning Test If the grid bars are of a color other than the subject, and the detected subject appears to be broken in multiple parts, use the

Dilation filter. Increase the value near Dilation until the yellow body

is entirely over the subject's body. After that, reduce the size of the yellow blob by means of the second Erosion filter.

For details, see Advanced detection settings: Subject contour in the

EthoVision XT Help.

Minimize the effect of the subject's shadow

Make the contrast range smaller, to exclude the pixels in the shadow. For example, if the subject has dark fur, and the shadow is included in

the yellow blob, then next to Dark move the left slider to the right. This

will exclude smaller values of contrast. You should see the yellow area over the shadow shrinking. EthoVision XT will not follow the shadow anymore. Do this in small steps, until the shadow is no longer detected. Make sure that the whole body is still detected in all parts of the cage.

tip If the background looks too similar to the color of the subject, place

a sheet of paper or plastic in the drawer that you find under the shock grid. Try to make as much contrast as possible between the subject and the background.

Set Activity analysis for freezing detection

In the Detection Settings window, click Activity. The Fear Conditioning Test Adjust the settings in such a way that a large number of purple pixels appear only when the subject moves.

If the Activity threshold is too low, purple pixels also appear in the

background (see the next figure, left and middle).

Increase the value of Activity threshold until you see the purple

pixels appearing only when for example the mouse moves its head (see the next figure, right).

If the Activity threshold is too high, purple pixels disappear

completely. That's not good either, because you need to have a sufficiently wide range of the amount of purple pixels in order to detect immobility (freezing). When the subject freezes, the number of purple pixels should be minimized.

For more information

See Activity settings in the EthoVision XT Help.

Click the video button next to Activity, and watch the video

tutorial.

For details about the advanced detection settings, See Configure

Detection Settings in the EthoVision XT Help. The Fear Conditioning Test

troubleshooting

All the FCS devices are listed for Arena 1 only. For the other arenas, all

is set to . This may have occurred when you selected the number of arenas (for example, 4) after defining the COM ports for the FCS cages. If, at the time of defining the COM ports, the number of arenas was still 1, EthoVision XT assumes that all the devices must be associated with one arena, that is, Arena 1. Do the following: 1. In the Arena-Hardware Mapping window, select each row and click Remove device. Do so to remove all the devices. 2. Choose Setup > Experiment Settings. 3. Under Trial Control Hardware, click Settings, then choose Ugo Basile

F.C. Controller. In the window that opens, make sure that the COM

ports are listed and click OK. 4. Under Number of Arenas, select the number of arenas you work with. 5. Choose Setup > Arena Settings and click the Arena-Hardware

Mapping button. At this point the devices should be correctly

selected under one of the arenas. The Fear Conditioning Test

Acquire the data

things to check first

Before starting one or more trials, it is useful to do some checks.

Hardware

Check that all devices are properly connected according to the schemes in this manual. Check that all devices are powered up. Insert the conditioning cage in the cubicle as in the figure below. The cage fits between the two clear support angles attached to the IR light box.

Test the devices In EthoVision XT

Choose Setup > Arena Settings. Open the Arena Settings you want

to use. Click the Arena - Hardware Mapping button and test the

devices (page 203). The Fear Conditioning Test

Run a test trial

To check that the system works correctly, run a test trial with a basic Trial Control settings profile that contains actions and conditions.

After data acquisition, in the Analysis profile under Hardware

choose Hardware command and define the event you want to view,

for example Device A, Output 1 High (=Light on).

Choose Analysis > Results > Integrated Visualization and check

whether the event occurred at the expected time.

acquire data

In EthoVision XT, choose Acquisition > Open Acquisition. Follow the general procedure described in the EthoVision XT Help. Browse to Acquire Data > Acquire one trial. The Fear Conditioning Test

Analyze the data

basic steps

1. Define the intervals you want to analyze. Choose Analysis > Data Profile > New. See analyze intervals below. If you want to calculate statistics for the whole duration of your trials, you can skip this step.
2. Choose Analysis > Analysis Profile > New. Choose the variables you want to analyze. For example, velocity, time in zone, or activity. See Dependent variables below.
3. Choose Analysis > Results > Statistics and Charts to calculate

statistics, or Integrated Visualization to view the video and the

variable plotted against time.

analyze events

Add the following variables, if not already listed in the Analysis profile:

Activity state to analyze freezing events. For more information

about Activity and Activity state, and how to set the thresholds to detect freezing, see the EthoVision XT Help. Distance moved, and Movement to quantify locomotory behavior.

Hardware command especially to visualize events like Light On,

Shock On, etc. together with the video and the tracks (Analysis >

Results > Integrated Visualization).

Trial Control Event to visualize for example when a condition

becomes true. Use this especially for testing purposes. The Fear Conditioning Test

analyze intervals

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). The Fear Conditioning Test 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. In the following example, we 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. See the figure below.

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 showed in dimmed colors. Check that the intervals defined correspond to the time segments that you want to analyze. The Fear Conditioning Test In the example below, 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 also the Chapter Conditioning tests in this manual. The Fear Conditioning Test

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 Top light LxWxH: 28.6 x 29.3 x 8.5 cm (11 17/64" x 11 17/32" x 3 11/32") Controller LxWxH: 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 CE marking: see the next page The Fear Conditioning Test

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