VIDEO FEAR CONDITIONING
Contents
1. Original data type Choose the file type that best describes your data Characters such as commas or tabs separate each field Fixed width Fields are aligned in columns with spaces between each field T le o esee Ni Li N I El n Start import at row 1 Fe File origin 437 OEM United States o alal Preview of file Vfreeze screen shots for vfc Amygdala Data csv Date Exported 03 09 05 10 33 45 Run Time 0 seconds xperiment VF030905 095732 sh rial Trail 1 i geo a eas es www NB NS o o IR IR IR ID N Nja 6 6 04M Wi 22 de gts M 4 MN Sheet1 Sheet2 Sheet3 lt gt Cancel Next gt Einish Rea NUM Click Next and progress through steps 1 3 and then Finish When the import is complete the motion index data in rows appear as a function of time in columns Time increases in Column A and the corresponding data for each chamber appear in each row chambers 1 4 left to right CHAPTER 11 Component Analysis One may choose to compare PERCENT FREEZE across different events within a single experimental session For instance if several conditioned stimulus unconditioned stimulus CS US pairs are presented within a single session then the amount of time the subject spends motionless during each presentation of each CS may provide meaningful information on the rate of conditioning Figure 11 1 Data from Four CD 1 Mi2. To add a new configuration to the list click Add The screen shown in Figure 4 4 will appear proceed to step 4 To exit this screen click Exit To continue with camera calibration proceed to step 6 Figure 4 4 Adding a New Configuration Configuration Setup Box Name Not Assigned Connected VFC 100 Offset E Bit White House Light NIR House Light ooooooocoo ooooooooo O O O O O 0 O O O 4 Create a new configuration by entering the desired box name into the Box Name field Box refers to an individual experimental setup and includes the Sound Attenuating Cubicle SAC video camera conditioning chamber and all stimuli associated with that chamber The hardware settings including camera settings and stimulus addresses port offset bit must be set for each Box in the system 5 Next select a camera from the Camera pulldown menu Then enter the VFC 100 Offset see step 7 for details on the VFC 100 Offset Once all of the desired selections have been made select Ok The screen shown in Figure 4 3 will appear again 6 Select the camera to calibrate from the Configuration Manager screen Figure 4 3 by either double clicking on the Id of the desired camera or by highlighting the desired camera and clicking Edit The screen shown in Figure 4 5 will appear Figure 4 5 Configuration Setup Configuration Setup Box Name Camera Workstation 1 VFC 100 Offset D Stimulus Sound Stim Light 1 0
3. EUM Total Avg Min Max Component Freeze Time Time Component Motion Motion Motion Group Name Cnt Freeze Freeze Time Freeze Index Index Index 2 CS 1 42 1 433 0 12 2 39 438 28 0 2471 2 CS 2 30 1 233 0 1 2 06 418 33 0 1998 2 CS 1 52 23 767 1 98 39 61 235 15 0 2417 2 CS 2 42 17 533 1 46 29 22 17 23 0 1986 csv Dependent Variables The csv file contains identifiers and component analysis details as well as four dependent variables listed below 1 Freeze Count or Freeze Episodes Number of freezing events as defined by either the linear or discrete method of observation respectively during the component 2 Time Freeze Total number of seconds the subject spends motionless during the component interval 3 Percent Total Time Freeze The amount of time the subjects spends motionless during the session divided by the total amount of time for the session 4 Percent Component Time Freeze The amount of time the subject spends motionless during the component divided by the total amount of time within that component 5 Average Motion Index The average motion index during the component 6 Minimum Motion Index The minimum motion index during the component 7 Maximum Motion Index The maximum motion index during the component Batch Component Summary Report To simultaneously apply a cmp file to multiple raw data files go to File Reports Batch Component Summary This option will allows the user to view and
4. Freeze Duration is set to 15 frames then the subject must freeze for 2 a second before the event is recorded by the software as a freeze If the Sample Rate is 3 fps and the Minimum Freeze Duration is 3 frames then the subject must remain immobile for an entire second to register as a freeze In Figure 7 1 each filled in circle represents a video frame 30 fps The subject is frozen at 0 2 seconds because movement is below the Motion Threshold and remains frozen until 0 75 seconds Using the linear method the first Freeze Episode is recorded at 0 4 seconds because the Minimum Freeze Duration 6 frames has expired Movement is recorded at 0 75 seconds and the subject is immobile again at 0 95 seconds Once the Minimum Freeze Duration has expired at 1 15 seconds the second Freeze Episode is recorded Note that the Motion Threshold must be exceeded before a second Freeze Episode can be recorded therefore when using the linear method Freeze Episodes must be separated by periods of movement Figure 7 1 Description of the Linear Method Linear Analysis 100 80 60 Motion Index 40 20 Motion Threshold 0 0 0 2 0 4 0 6 0 8 1 0 1 2 1 4 Minimum Freeze Duration Time sec Parameters Data Sample Rate 30 frames per second Freeze Episodes 2 Session Time 1 4 sec Freeze Duration 0 833 sec 25 frames Motion Threshold 10 Percent Freeze 59 5 Minimum Freeze Duration 6 frames 0 2 sec Discrete Method Th
5. HAR CAMERA MINI HUB Hardware Guide Description Sound Attenuating Cubicle SAC Conditioning Chamber Interface Cabinet with 28 VDC PCI Interface Auditory Stimulus Output Cards PCI Interface Card Aversive Stimulator Quick Disconnect Harness Passive Connection Panel Stimulus Light with Diffuser Video Camera with NIR Filter Light Controller Light Panel Fan Repeater Fire Wire Hub with Inverter Quantity One per system One per SAC One for every four Chambers One per Interface Cabinet One per Chamber One for every four Chambers One per Chamber One per Chamber One per Chamber One per Chamber One per Chamber One per Chamber One per Chamber One per Chamber One for every two Cameras Location Inside the Cubicle Outside the Cubicle SG 6080D Interface Cabinet SG 6080D Interface Cabinet Computer Outside the Cubicle Back of Chamber Inside the Cubicle Inside the Chamber Left Cubicle Door Outside the Cubicle On top of the Cubicle Inside the Cubicle Outside the Cubicle MED ASSOCIATES INC Cable Guide Figure 1 12 DIG 700C Ribbon Cable Figure 1 13 SG 210CP 2 Pin Molex Cable Figure 1 14 SG 210CB 25 Serial Port Cable Figure 1 15 SG 210G 10 DB 9 Cable VIDEO FEAR CONDITIONING MED ASSOCIATES INC Figure 1 16 SG 216A 10 3 Pin Molex Cable Figure 1 17 VDO CAB 15 Fire Wire Cable Figure 1 19 NIR 102 Light Control Interface Cable VIDEO FEAR CONDITIONING MED ASS
6. Session describes the options pertaining to dependent measures The VFC System is entirely automated and records video on a PC hard drive Digital video cameras are used to simultaneously capture data from up to four fear conditioning chambers mice or rats The Video Freeze run time display provides information on current CS US durations and inter trial intervals as well as displaying the video input while a session is being conducted see the section on Recording the Session for more details All data are stored to the hard drive and can be analyzed using the Video Freeze Data Analysis Utility refer to the section on Analyzing raw Data in Video Freeze Use this data analysis utility to simultaneously view the video and quantitative results The data analysis user interface allows researchers to focus on specific intervals within any given trial for detailed examination of the results Med Associates is dedicated to providing the scientific community with reliable and innovative behavioral testing tools Please contact technical support at support med associates com with questions regarding the Video Fear Conditioning System Med Associates welcomes other comments or suggestions on how to improve this product Thank you for purchasing the Video Fear Conditioning System MED ASSOCIATES INC VIDEO FEAR CONDITIONING Independent Variables The Video Freeze Software allows researchers to define stimulus intensities and durati
7. SvIbv AN2 40 AIF SUOIJJBUUO sn nulljS 9AISI9AV 0809 5S 40 1U014 a 001793 pan 001 241 001 93A 9 qe uoqqiy 9002 910 Y 1Nd1NO X08 1NdLNO xoa LIILIIIIIJ CZ i J9 49AU pepi OJd BHulsn 139p no JIEM OL Y i qnH M 2114 9Iqe5 M ST qV2 OQdA 9 q85 21IM9114 21Buy 3451y Vu d9 WE I 3HIJ 8V2 PIED IOdb0Z 9IQ pue SpJe5 9JIM 2114 YUM Je3nduio 5 1210 IEM OL Suonoeuuo e2eJJo1u 10 MED ASSOCIATES INC VIDEO FEAR CONDITIONING Step by Step Instructions 1 Using the DIG 700C Ribbon Cable connect the DIG 700F PCI Interface located in the SG 6080D cabinet to the DIG 704PCI card located on the back of the computer DIG 700C Ribbon Cable DIG 704PCI Interface Card 7 Back of DIG 700F 1 Computer Interface Card SG 6080D Cabinet 2 Using an SG210 CP 2 2 pin Molex cable connect the 28V port on the DIG 700F PCI Interface located in the SG 6080D cabinet to anv available 28V port on the rear of the SG 6080D cabinet SG 6080D OQ Q o H DIG 700F POWER IN Interface Card An 110 Volts N H Fuse 8 Amp 250V MINI FB H SG 210CP 2 i Back of oO Fi Computer oc med ASECCISTRE INU Back of SG 6080D Cabinet 11 MED ASSOCIATES INC VIDEO FEAR CONDITIONING 3 Using an SG 210 CB 25 25 serial port cable connect each VFC 100 stimulus output card located in the SG 6080D cabinet to the corresponding VFC 716 Passive Co
8. is because several Freeze Counts can occur without a record of movement using the discrete method Observation Interval and Duration must be set to greater than 1 fps and the Observation Duration must be less than the Observation Interval The Observation Interval defines the time period between the beginning of one observation and beginning of the next Therefore with a Sample Rate of 30 fps and an Observation Interval of 15 there will be two observations per second Figure 7 2 Description of the Discrete Method Discrete Analysis 100 80 60 40 Observation Observation Duration Interval 20 Motion Threshold Motion Index de ATI nd l WW Ww rad didit ee 0 0 0 2 0 4 0 6 0 8 1 0 1 2 1 4 Time sec Parameters Data Sample Rate 30 frames per second Freeze Counts 4 Session Time 1 4 sec Total Counts 7 Motion Threshold 10 Percent Freeze 57 1 Observation Interval 6 frames 0 2 sec Observation Duration 3 frames 0 1 sec Select Save to save this experimental configuration for later use ASSUCIATI COS INC V CHAPTER 8 Video Capturing System Calibration PRIOR TO LOADING THE SUBJECTS INTO THE CHAMBERS the video capturing system in each chamber must be calibrated Click the Calibrate button on the main screen indicated with a yellow arrow in Figure 8 1 Ensure that the doors on all the sound attenuating cubicles and conditioning chambers are closed prior to calibrating Figure 8 1 T
9. left then the right doors The vertical seam between the two doors should be evenly spaced from top to bottom as shown in Figure B 3 If they are not proceed to Step 4 Figure B 3 Front View of SAC with Even Vertical Door Gap Door Gap Even at Top and Bottom 4 Adjust the door position using the Horizontal Adjustment Screw Turn the screw clockwise to move the door closer to the vertical center of the SAC and counterclockwise to move it further For example if vertical gap between the doors is smaller at the top of the SAC than at the bottom assess which door needs to be adjusted If it is determined that the top of the right side door is too close to the center then the Horizontal Adjustment Screw on the top hinge should be turned counterclockwise Figure B 4 Horizontal Adjustment Screw Horizontal Adjustment Screw APPENDIX C Camera Filter Care and Handling Instructions Proper care and handling of the camera filter are crucial to the proper operation of the system Clean the filter gently only if necessary Loose particles should be removed with a bulb puffer or with a filtered pressurized air cleaner If necessary gently wipe the surface using anhydrous alcohol and lint free lab towels Use a new surface of the towel with each wipe Avoid touching or wiping the filter with bare fingers MED ASSOCIATES INC VIDEO FEAR CONDITIONING APPENDIX D Installing Contextual Inserts A Frame Insert Begin installation of
10. the NIR 100 Light Controller to the OUTPUT 7 connector on the VFC 716 VFC 716 OUTPUTS INPUTS 15 to 28V P CM ss NIR Light WhiteLight Tees 2 see Control Input Control Input To Light Panel 3 eee 3 eee 4 eee 4 bee 5 ses VFC 716 NIR 102 Back of NIR 100 Light Controller stess Passive Connection All cables running into the Sound Attenuating Cubicle SAC should Tan Panel be run through the grommet plate on the right side of the SAC DE 28 Volts A EE 16 Using another NIR 102 cable connect the NIR Light Control Input on the rear of the NIR 100 Light Controller to the OUTPUT 8 connector on the VFC 716 VFC 716 _ OUTPUTS INPUTS 15 to 4 28V d HH Oe NIR Light WhiteLight a Control Input Control Input To Light Panel VFC 716 Passive Connection All cables running into the Sound Attenuating Cubicle SAC should Panel ee be run through the grommet plate on the right side of the SAC DEI 28 Volts A SEE A NIR 102 Back of NIR 100 Light Controller 17 Using the NIR 101 cable connect the To Light Panel connector on the rear of the NIR 100 Light Controller to the only connector on the NIR 100R Light Panel 15 to 28V t9 Bl UE ECE Cai NIR Light WhiteLight Control Input Control Input To Light Panel Back of NIR 100 Light Controller NIR 100R Light Panel Top of SAC 18 MED ASSOCIATES INC VIDEO FEAR CO
11. the A Frame contextual insert by sliding the chamber forward inside the SAC and opening the chamber door Using the handle pull the grid floor and waste pan forward until they are nearly removed from the chamber Insert the A Frame Contextual Insert so that there are two grid rods visible on either side when viewed from above Refer to Figure D 1 Figure D 1 A Frame Contextual Insert Installed 1 l n iL 1 U i x SR LEE Smooth Floor Insert Install the smooth floor contextual insert by sliding the chamber forward inside the SAC and opening the chamber door Using the handle pull the grid floor and waste pan forward until they are nearly removed from the chamber Place the smooth floor insert over the grid floor Refer to Figure D 2 Figure D 2 Smooth Floor Contextual Insert Installed 57 MED ASSOCIATES INC VIDEO FEAR CONDITIONING Curved Wall Insert Begin installation of the curved wall contextual insert by sliding the chamber forward inside the SAC and opening the chamber door Using the handle pull the grid floor and waste pan forward until they are completely removed from the chamber Place the insert inside of the chamber so that the ends are braced against the side wall supports of the chamber Refer to Figure D 3 Be sure to reinstall the grid floor and waste pan prior to placing animals in the chamber Figure D 3 Rounded Back Wall Contextual Insert Installed 58
12. the VFC 716 Passive Connection Panel VFC 716 OUTPUTS INPUTS Ix Man Ces ca sss ses see coo Ton Es Eaa Ten ler DEP er A be VFC 716 Passive Connection Panel SG 216A 10 Cable i NOT FOR HUMAN USE 50 ka DUMMY il WG fi MED O GRID OUTPUT Control LOAD Vour o POWER IN CAUTION L Y g x a A 110 Volts VAN HIGH VOLTAGE Z TTLiSwitch Closure Fuse 0 5 Amp SB Back of ENV 414S Aversive Stimulator All cables running into the Sound Attenuating Cubicle SAC should be run through the grommet plate on the right side of the SAC 6 Connect the ENV 227M Stimulus Light with Diffuser on the left side of the Conditioning Chamber to the OUTPUT 1 connection on the VFC 716 Passive Connection Panel 13 MED ASSOCIATES INC VIDEO FEAR CONDITIONING 7 Connect the speaker to the input on the side of the VFC 716 Passive Connection Panel E s HEGRE ERE 8 Connect the fan inside the Sound Attenuating Cubicle to one of the three 28V connections on the VFC 716 Passive Connection Panel y e e E a ao B BEBE S 14 MED ASSOCIATES INC VIDEO FEAR CONDITIONING 9 Using an SG 210CP 25 2 pin Molex cable connect any available 28V connector on the VFC 716 Passive Connection Panel in Chamber 1 to a 28V connector on the rear of the SG 6080D cabinet VFC 71
13. values in the Function Detail area of the screen or by pressing and holding the Ctrl key while simultaneously clicking and dragging the ends of the stimulus representation Any changes made to the properties using the mouse will be displayed in the Function Detail area of the screen In Figure 5 4 the first representation of Stimulus 1 has been selected The Function Detail area of the screen is also where other Stimuli parameters are defined as shown in Figure 5 4 The Volume dB Frequency Hz and Rise Time ms can be changed as well as whether the audible stimulus is a Tone or Noise In the example shown in Figure 5 4 Stimulus 1 begins 10 seconds after the experiment starts and turns off 30 seconds later Stimulus 2 begins 30 seconds after the experiment starts and the duration 10 seconds All stimuli are independently defined Figure 5 4 Editing Protocol Setup Protocol Setup Function Detail Total Time s AMLOZE Interface Start Time s 10 Diss m Duration s 30 Noise volume Db 90 Frequency Hz 2000 Rise Time ms al amp e ES Setup Stimulus Stimulus 1 Function Add Stimulus 2 Delete Protocol New Load Save Save as kem Saving a Protocol Once all of the desired stimuli have been added to the protocol select Save As to save the protocol and all stimulus properties Save the protocol to the desired directorv it may be useful to cre
14. window should contain a clear bright image of the empty Fear Conditioning Chamber If the image is fuzzy the focus of the lens may need to be adjusted Refer to the next section for camera focusing instructions VAT rs A COCOA PP A FI co THIS LUITA FA f AT ff TX RIT TT TC AIT A Vani VIEN ASC A CC N NEO FEAD CN N T TC N NC MCU ADOULIAIED INC VILLEU TLAN CUNYVILILUINING CHAPTER 5 Protocol Setup Customized experimental protocols can be efficiently designed and implemented using the Protocol Setup utility of Video Freeze software This utility creates a pro file that is later executed when conducting an experimental session Select the Video Freeze icon on the computer desktop and select File Protocol Setup Stimulus conditions and session duration are defined in Protocol Setup Defining the Stimulus Conditions Select File Protocol Setup and the following screen will appear Figure 5 1 Opening the Protocol Setup Window Protocol Setup Function Detail Total Time s ag Setup E Function Protocol To define the stimulus conditions click New The Stimulus Definition screen will appear Set the Total Time which is the total duration of the protocol and select the Stimuli to be used in the protocol by checking the corresponding boxes Note that only the Stimuli defined in Stimuli Definition Chapter 3 appear on this list When the desired stimuli have been selected clic
15. 0 0 0 0 0 0 0 White House Light Calibrate Camera 7 Choose a camera from the Camera pull down menu and enter the offset in the VFC 100 Offset field The VFC 100 offset refers to the hardware address of the VFC 100 card Determine which Box that each VFC 100 is connected to and refer to Table 4 1 to determine the appropriate offset numbers for the default configurations Enter the VFC 100 offset and click OK Table 4 1 Default Stimulus Configuration Settings Boxi Box2 Box3 Box4 ANL 926 sound Port 790 790 790 790 Offset 0 1 2 3 Bit 0 0 0 0 DIG 704 I O General Port 792 792 792 792 Offset 0 2 4 6 Stimulus Light Bit 1 1 1 1 Aversive Stimulus Bit 2 2 2 2 Once all of the settings are correct click on Calibrate Camera The following screen will appear Figure 4 6 Calibrate Camera Calibrate Video Display Window Average Intensity Standard Deviation Brightness Camera Controls 500 1000 Gain M 50 100 150 200 250 Shutter i 1000 1500 l 300 Grayscale Histogram Display Average Grayscale Intensity Indicator 8 Notice that initially the video image in the Video Display Window is totally black Figure 4 6 Move all of the green Camera Control sliders all the way to the left 9 Begin adjusting the camera settings by moving the Brightness slider to the right As the Brightness slider is moved back and forth a single narrow peak will move back and forth in the G
16. 00 01 30 000 00 01 45 000 NOTE The video cameras are extremely sensitive to movement Therefore any vibration in the laboratory environment may register as movement in the Motion Index A f Al rU CHAPTER 10 Data Analysis Types of Data Files When an experiment is conducted the Video Freeze Software simultaneously creates three different types of data files One is a video file wmv that can be viewed using Window s Media Player see Figure 10 1 Another is a summary file txt that can be exported to any spreadsheet program such as Excel see the section on Exporting Data to a Spreadsheet The third type is a raw data file that can only be viewed using Video Freeze This raw file contains the motion index data see Figure 10 3 See the next section to learn how to use Video Freeze to re analyze the raw data file Figure 10 1 Windows Media Player File wmv C windows Media Player Bm Now Playing 8 ow Playing List Total Time 2 00 VF030905 095732 Trail 1 Figure 10 2 Summary File txt Date Printed Run Time 03 09 05 10 23 34 null Protocol Cs Multiple CS US trials session pfo Experiment t VF031505 101635 Trial 2 d Video Saved Ves Sample Rate fps 30 000 Motion Threshold au 20 Detection Method Observation Interval Observation Duration Number of boxes Discrete 15 frames 10 frames 1 00 00 00 00 Box d Group Amvgdal
17. 00 150 Shutter R 300 1000 12 Now increase the Gain slider bv moving it to the right until the right edge of the Gravscale Histogram is at the right edge of the Gravscale Histogram Displav Notice that the Gravscale Histogram now contains jagged bumps Figure 4 10 Spreading the Gravscale Histogram by Increasing Gain Calibrate Jagged Bumps on Histogram Histogram Edge Spread to Right Average Intensity Standard Deviation 0 064 Brightness 1 I l I Increase Gain E us O SO 100 150 200 250 l 1 1 I I 300 1000 1500 MED ASSOCIATES INC VIDEO FEAR CONDITIONING 13 To eliminate these jagged bumps decrease the Gain slightly This will also result in a gap at the right edge of the Grayscale Histogram Display Figure 4 11 Smoothing the Histogram Curve by Decreasing Gain Calibrate Smooth Histogram Curve Gap on the Right Average Intensity Standard Deviation oos Brightness Decrease Gain 1000 1500 14 Increase the Shutter control by moving the slider to the right This will spread the histogram out so that it s right edge is at the right edge of the Grayscale Histogram Display Figure 4 12 Spreading the Histogram by Increasing Shutter Calibrate Smooth Histogram Curve Histogram Edge Spread to the Right Average Intensity Standard Deviation 0 069 Brightness f RE 1000 Gain m li Save 100 150
18. 200 250 Increase Shutter EET 1560 I I I I 1000 1500 The grayscale histogram should be fairly smooth with a clear peak near zero black and several broad peaks in the mid and upper ranges various shades of gray Note that the peak near zero black will be more or less apparent depending upon the view of the camera In the above example the black handle on the grid floor is visible in the camera view This handle accounts for the presence of the black peak in the grayscale histogram If this handle is not in the camera view the peak near zero will be less obvious and may even be totally absent The key is to adjust the camera settings to make maximal use of the camera s dynamic range This is accomplished by setting the camera so that the right and left edges of the grayscale histogram are just reaching the edges of the histogram graph NOTE It is important to note that the steps above are MEANT TO BE USED AS A GUIDELINE and that fine tuning may still be necessary The Brightness Gain and Shutter may need adjusting based on the color of the animal Fine tune the system with the animal in the chamber until a satisfactory motion index is attained Figure 4 13 Grayscale Histogram Characteristics of Properly Calibrated Camera Smooth Histogram Curve Right Edge of Histogram at Right Edge of Graph Left Edge of Histogram at Left Edge of Graph Average Intensity Average Grayscale Value in 130 s Now the Video Display
19. 6 0 IPUTS INPUTS se 1 eee 2 eee 2 eee 3 eee eee 4 0s0j 4 eos 5 see e ses 7 see eee SG 6080D el O ARENA B Fuse 8 Amp 250V o MINI FB O SG 210CP 25 oA eee 19 VFC 716 Passive Connection Panel DE 28 Volts o Ho Back of SG 6080D Cabinet MEd associates inn All cables running into the Sound Attenuating Cubicle SAC should be run through the grommet plate on the right side of the SAC 10 If there are multiple chambers then an SG 210CP 25 cable should be used to connect a 28V connector on the VFC 716 in Chamber 1 to a 28V connector on the VFC 716 in Chamber 2 then another cable from Chamber 2 to 3 then another cable from Chamber 3 to 4 Chamber 1 Chamber 2 Chamber 3 Chamber 4 VFC 716 VFC 716 VFC 716 VFC 716 g OUTPUTS INPUTS e g OUTPUTS INPUTS e a OUTPUTS INPUTS e a OUTPUTS INPUTS e ifesej 1 boe ifesej 1 eee ifesej 1 bee 1fece eee 2 eee eee 2 eee 2 eee 2 eee 2 boe 2 eee eee 3 eee 3 eee 3 eee 3 eee 3 eee 3 eee 3 eee 3 eee 4 eee 4 bee 4feeej 4 eee 4 eee 4 boe 4 eee 4 boe 5 eee 5 eee 5 eee 5 eee 6 eee 6 eee 6 eee 6 eee 7 eee 7 ees 7 eee 7 ees Power 8 esej 8 eee 8 eee 8 eee pr E Bb Eh Ei 28 Volts 28 Volts 28 Volts 28 Volts ZNG
20. DANIS eme A gt ge A gt lt A DUN l l Il All cables running into the Sound Attenuating Cubicle SAC should be run through the grommet plate on the right side of the SAC 15 MED ASSOCIATES INC VIDEO FEAR CONDITIONING 11 Using the VDO CAB 15 Fire Wire cable connect one of the Fire Wire cards on the back of the computer to one of the Fire Wire hubs Fire Wire Cards Back of Computer Fire Wire Hub 1 12 Using the other VDO CAB 15 Fire Wire cable connect the second Fire Wire card on the rear of the computer to the second Fire Wire hub Fire Wire Cards Back of Computer Fire Wire Hub 2 16 MED ASSOCIATES INC VIDEO FEAR CONDITIONING 13 Using a CAB FIRE I 3M 6P RA Right Angle Fire Wire cable connect each camera to a Fire Wire hub so that there are two cameras connected to each hub The right angle connector goes to the camera refer to Figure 1 5 CAB FIRE I 3M 6P RA Fire Wire Hub with Cameras Connected All cables running into the Sound Attenuating Cubicle SAC should be run through the grommet plate on the right side of the SAC 14 Connect the Fire Wire Inverters into each of the Fire Wire Hubs and plug in to a standard wall outlet B 00900 Fire Wire Hub Included Power Inverter 17 MED ASSOCIATES INC VIDEO FEAR CONDITIONING 15 Using a NIR 102 cable connect the White Light Control Input on the rear of
21. ERMY DOCUMENTSYSUB JECT 2 FINAL 2 File Experiment View Menus Help Component Report Component Definition Name Component Na Start Time Duration csi 00 00 30 0 30 000 Start Time S 3 O cs2 00 02 00 0 150 000 csminust 00 03 30 0 30 000 Add New CSminus2 00 05 00 0 30 000 Duration s 39 Fa OK Cancel 9154 763 7 610 458 x D TG fa E 8 o z 3054 Click Add New to add a component or OK when all the desired components for the analysis have been included As new components are added to the analysis each component will appear in the Report Component Definition display The component analysis cmp is a file that can be saved independently of any raw data file Choose Component Save and select the directory and filename for the component analysis see below This procedure has created a component analysis that can be applied to any raw data file MED ASSOCIATES INC VID EO FEAR CONDITIONING Figure 11 3 Saving a Component Analysis cmp file Lookin GJ My Documents amp ek Fav 3 E Mv Music E Single Trial US 1 txt LI My Pictures Single Trial US 1 wmv My Recent My Videos fiss 2 30 min 2 raw 5 30 min discrim Test 1 raw E ss 2 30 min 2 txt E E 30 min discrim Test 1 txt lss 2 30 min 2 wmv Desktop Documents 30 min discrim Test 1 wmv Ss 2 30 min discrim 2 wmv E 30 min discrimination test 1 raw 9 Subject 2
22. Experiment Id VFOZI6DS 155034 Trial D Load Save Protocol rl Save Video M Notes Select configuration and setup subjects For this experiment Configuration T Camera l Motion Threshold taut 18 Sample Rate Fps E Frs Method Linear Min Freeze Duration Frames JU 00 00 00 133 C Discrete Ok Cancel Experimental Setup The Experiment Setup screen includes the following fields menus Experiment Id If Auto Experiment ID Generation is selected on the Options menu see Figure 7 2 a filename will automatically appear in the Experiment window This filename will include the date and time of the experiment and follows the form VFMMDDYY HHMMSS This information can be altered to suit the particular experiment Figure 6 2 Auto Experiment ID Generation Fle Experiment R6 A w Auto Experiment Id Generation SA Ml me 4 ii Trial Enter trial session information in the Trial field Protocol Next to the Protocol title click the box labeled to open the protocol pro file saved earlier See Saving a Protocol Save Video Saves the video output as well as the quantified data NOTE Saving video substantially increases the amount of space required to save experimental data This is not a major limitation since the Video Freeze computer has a large hard drive 200 GB and a DVD burner Notes Enter any relevant information here such as instructions on how to complete the experi
23. NDITIONING 18 Using the included power cord plug the SG 6080D cabinet into a standard wall outlet SG 6080D O e Ne ANH MINI FB a o fo Back of SG 6080D Cabinet Fuse 8 Amp 250V H o med aSSOCMIES ING 19 Using the included power cord plug the ENV 414S Aversive Stimulator into a standard wall outlet NOT FOR HUMAN USE 50 K DUMMY f ii 0 MED GRID OUTPUT Control LOAD Vour 3 am POWER IN A CAUTION we 110 Volts VAN HIGH VOLTAGE TTL Switch Closure Fuse 0 5 Amp SB T Back of ENV 414S NOTE It is not necessary to apply power to the NIR 100 Light Controller via the 15 to 28 V DC connector Power is supplied via the Light Control Input connectors 15 to 28 ox NIR Light WhiteLight Control Input Control Input To Light Panel Back of NIR 100 Light Controller 19 MED ASSOCIATES INC CHAPTER 3 Getting Started Once the hardware and drivers are correctly installed the configured for each chamber Figure 3 1 Video Fear File Menu Experiment Options Help Opern Set Working Directory Stimuli Definition Camera Identities Chamber Configuraion Protocol Setup Extract Video Stream Reports d Exit VIDEO FEAR CONDITIONING software needs to be From the File menu select Stimuli Definition The following will appear Figure 3 2 Stimulus Names Stimulus Names Sound Stim Light Shocker Not Connected Not Connected Not C
24. OCIATES INC VIDEO FEAR CONDITIONING Figure 1 20 CAB FIRE I 3M 6P RA Right Angle Fire Wire Cable Table 1 2 Cable Guide Part Number Description DIG 700C Ribbon Cable SG 210CP 2 2 pin Molex SG 210CB 25 25 Serial Port Cable SG 219G 10 10 DB 9 Cable SG 216A 10 3 pin Molex CAB FIRE I 3M 6P RA Right Angle Fire Wire Cable VDO CAB 15 15 Fire Wire Cable NIR 101 Light Control Input Cable NIR 102 Light Control Interface Cable VIDEO FEAR CONDITIONING MED ASSOCIATES INC CHAPTER 2 Guide iring Prior to making any connections be sure that all equipment is turned off Failure to do so may result in equipment damage Quick Reference 4 Andino 01 91489 ZOT UIN gt 8 INdLNO 01 3 925 ZOT XIN jaueg 34517 HOOT UIN gt Q 01 21483 TOT YIN eueg 346517 HOOT HIN g3Sn LON OOT YIN JO J929 SUOIJIDUUO IUDIT 21429 SZ 820IZ DS 4 21q29 z d2017 5S d0809 DS Jo x224 nui sajar30888 PAU M OL QO8O9 DS JO Y2Eq uo 9 J0J99UU0 A8Z AUB 0 91989 SL AHIOTT DS TEE eloul e 211M 2114 Po JVS 242 Jo epis 3u861 y uo e3ejd JIWWOJ y U NOJUJ UNI eq pinoys DVS 2121qn2 Bulgenuazy punos y OU Buiuuni S9JQE2 Iv sanani sinaino guo4n LAdLNO 01 21489 gt OT VITE 9S Jequieu jo yoeg uo 4 SS9UJEH 100 4 PIO 0 21989 OT D6TC 9S jenno EM OL ind CET ISN NYHNH 401 LON O 0 ARNO ALES
25. VIDEO FEAR CONDITIONING VIDEO FREEZE SOFTWARE AN AUTOMATED ANALYSIS OF CONDITIONED IMMOBILITY SOF 843 User s Manual DOC 019 Rev 3 8 Copyright 2011 All rights reserved MED Associates Inc P O Box 319 St Albans Vermont 05478 WWW med associates com MED ASSOCIATES INC VIDEO FEAR CONDITIONING TABLE OF CONTENTS Chapel Licia 1 INUOGUGHOM a ee des aan aa ne add ile hi tions 1 Independent Variables ne nae nnne aeneae nnne nnn 2 Dependent Variables dns eines sata seen EIFE ei sie FEX ERES waa 2 HardWare CU e TNT 2 GADIE GUN M 7 CHapler 2 eee rr 10 WIFINd SUdeai ani 10 eTo AEE AE air ira 10 DLODDVSSEBD INSTrUCONS isirscerareneisrrorisirdtni rinri IE 11 Cite DUCK l vari garr Ar 20 Getting Started PPT A FID O in UNI EES 20 Cita DR nn 22 Camere melle e uio se ae eme ananas atea 22 AGIUSUNO Camera FOCUS erica DG ora c S 22 Calibration PrO CUTE samra a rt a A sia 23 CHD il g ane D een oc conne 30 protocol SEtt B RP MHOUO XOTM 30 Defining the Stimulus Conditions nnne meme nnn nnns 30 SV OS PIOLOGO urinaria 33 Modifying an Existing Protocol ss 33 CAD PO in eee RD coli 34 Starting an Experimental Session nsc nnne nne nan 34 Experimental Seb azera ara 34 Threshold Valles siria Ek v Edad varia dus naa 35 CNADier7 iii a 36 Methods Or ODSEV A HOM aaa aan a 36 Linear Meth PR 36 DI Crete MGTIO Gs TT E A EE EEEE 37 CAD
26. a Animal Animal 1 Total Time s Avg Motion Freeze Counts 120 000 00 500 00 333 seconds seconds Percent Freeze Analyzing raw data in Video Freeze Select File Open and locate the raw data file of interest The Video Freeze Data Analysis window provides the same information as the run time window video motion index subject identifying information freeze count etc Figure 10 3 The raw Data Analysis Window 7 Video Freeze File Experiment Help Animal Animal 3 Selected Box Cursors Boxi Box2 Time Range Delta X1 X2 00 00 00 33 X1 00 00 00 000 00 00 00 000 X2 00 00 00 000 Reanalysis Parameters Freezing Measures i 20 s Method Movement Threshold Recalc Avg Movement 70 4 e Linear Min Freeze Duration f 15 90 00 00 133 Freeze Count 7 Time Freezing 40 233 Discrete Percent Freeze 33 5 00 00 15 000 00 00 30 000 00 00 45 000 00 01 0 000 00 01 15 000 00 01 30 000 00 01 45 000 Use the stop play fast forward rewind tabs to play the video If modifying the Method Motion Threshold or Minimum Freeze Duration click on the Recalc button to calculate Freezing Measures To focus on a specific period of time during the session click on the blue timeline bar located below the motion index record Adjusting the blue bar by dragging the cursor causes use the motion index to expand The collapsed timeline bar represents the proportion of time out of the total ses
27. ata analysis CHAPTER 7 Methods of Observation Linear Method The dependent measures resulting from the linear method are im Percent Freeze time immobile total session time The run time window see the section on Recording the Session of Video Freeze also provides Instantaneous Percent Freeze time spent immobile current session time Cumulative Percent Freeze time spent immobile total session duration This percent freeze score will increase as the session progresses because time immobile will increase relative to total session time Cumulative Percent Freeze will approach Instantaneous Percent Freeze as the session progresses 2 Freeze Episodes number of freezing events defined by Motion Threshold and Minimum Freeze Duration 3 Freeze Duration total amount of time the subject spends immobile Minimum Freeze Duration number of frames When a subject freezes during the session and the motion index falls below the Motion Threshold the Minimum Freeze Duration is the temporal criterion that defines whether or not a Freeze Episode is recorded Minimum Freeze Duration is an important variable because it operationally defines a freeze The subject might momentarily stop moving but this immobility may not actually represent a freeze To prevent these immobility events from being recognized as Freeze Episodes set the Minimum Freeze Duration to equal to or greater than 1 If the Sample Rate is 30 fps and the Minimum
28. ate a folder titled Video Freeze Protocols The program will automatically save the file as a pro extension Figure 5 5 Saving a pro File to a Specified Directory Save Protocol as Save in Local Disk C m Documents and Settings 2 Program Files My Recent RECYCLER Documents jSystem Recovery 3 System Volume Information WINDOWS Desktop My Documents My Computer My Network Save as type Protocols pro Modifving an Existing Protocol To modify an existing protocol go to File Protocol Setup and then select Load and choose the desired protocol Click on a stimulus representation to modifv the properties of the stimulus Properties mav be modified either bv changing the values in the Function Detail area of the screen or by pressing and holding the Ctrl key while simultaneously clicking and dragging the ends of the stimulus representation Any changes made to the properties using the mouse will be displayed in the Function Detail area of the screen In Figure 5 4 the first representation of Stimulus 1 has been selected Click on a stimulus representation and click Delete to remove it from the protocol MED ASSOCIATES INC VIDEO FEAR CONDITIONING CHAPTER 6 Starting an Experimental Session From the main Video Freeze menu choose Experiment Start and the Experiment Setup screen shown below will appear Figure 6 1 Experiment Setup Experiment Setup
29. ates the driver and software installation was completed at the factory If the computer was not purchased from MED Associates follow the instructions to install the hardware drivers and software programs Before beginning the installation phone fax or e mail Med Associates with the registration information in order to receive the software installation password This password will be necessary during the installation process Insert the Video Freeze CD into the CD ROM drive and the screen shown in Figure A 1 will appear Click Install Video Freeze and the screen shown in Figure A 2 will appear Figure A 1 Installation Screen Video Freeze med ASSOCIATES INE Welcome to Video Freeze Install Video Freeze Extras Browse the CD MED ASSOCIATES INC VIDEO FEAR CONDITIONING Begin installing the drivers and software by clicking Install Complete the steps to install the drivers and software entering the desired User Name and Company as well as the password when prompted Successful installation of each item will be indicated by a green check mark and a red X will indicate an unsuccessful installation Once the drivers and software have successfully been installed the screen shown in Figure A 3 will appear Figure A 2 Video Freeze Installation Checklist Video Freeze Install Wizard med ASSOCIATES INE Welcome to the install wizard for Video Freeze The wizard will install Video Freeze on your computer To continu
30. ce During Fear Conditioning Comparison of US Intensity 1004 90 80 4 705 60 50 4 40 30 20 10 e 0 45 ma m 0 27 ma Percent Freeze Im NU 3 4 5 CS Presentations Oo The session was 30 min long with six presentations of a 30 s tone and 10 s aversive stimulus stimuli co terminated Two subjects received 0 45ma aversive stimulus and two received 0 27ma of aversive stimulus Percent freeze during the first 20 seconds of each CS presentation is illustrated above Component Summary Report To begin open a raw data file and select View Component Setup The Start Time and Duration of each event or component are user defined Therefore one can compare percent freeze across any interval within a single experimental session The data in Figure 11 1 reflects percent freeze across several CS US presentations within a session One can also use the component analysis to compare mean percent freezing during CS presentations and mean percent freeze when no discrete stimuli are present To define each component select Component Add see Figure 11 2 The Component display should appear Name each component and enter the Start Time s and the Duration s Percent freeze will be calculated for each component and the Start Time and Duration of the component determine the parameters for the calculation Figure 11 2 Component Definition Display VFreeze C DOCUMENTS AND SETTINGSYVFREEZ
31. e click Install Fire Wire Camera Driver DIG 704 Driver Med Test Video Freeze instrumentation and software for research Cancel Driver and software installation has been successfully completed Click Finish to close this screen Figure A 3 Customer Information Screen Video Freeze Install Wizard med ASSOCIATES INC Install Wizard Complete The Install Wizard has successfully installed Video Freeze Click Finish to exit the wizard Fire Wire Camera Driver y DIG 704 Driver 4 Med Test 4 Video Freeze instrumentation and software for research Finish 53 APPENDIX B Door Hinge Adjustment The steps in this section should be followed to ensure that the Sound Attenuating Cubicle SAC doors are properly adjusted 1 Close the left door and verify that it is properly sealed against the SAC When the door is properly sealed the gap between the door and the SAC is equal at both ends as shown in Figure B 1 If this gap is not even proceed to Step 2 Figure B 1 Top View of SAC with Properly Sealed Door Door Gap Even at Both Ends 2 Loosen the hinge retaining screw shown in Figure B 2 and slide the hinge until into the proper position Once the door is sealed correctly tighten the hinge retaining screw Repeat this step on the right door It may take more than one attempt to achieve an acceptable seal Figure B 2 Hinge Retaining Screw Hinge Retaining Screw 3 Close the
32. e discrete method is designed to mimic manual methods of monitoring freezing behavior The standard manual method of tracking freezing involves visually scanning each fear conditioning chamber at regular Observation Intervals e g every 4 seconds and observing behavior for a specified duration of time Observation Duration e g 1 second If the subject in the chamber is freezing for the entire Observation Duration 1 second a Freeze Count is recorded for that event If the subject is mobile during any portion of that observation period then no Freeze Count is tallied Therefore the number of counts recorded by the end of the experimental session serves at the quantitative measure of freezing Therefore the discrete method yields 1 Freeze Count number of freezing observations 2 Percent Freeze number of freezing observations total number of possible observations Observation Interval amp Duration number of frames Observation Interval and Duration are used to determine whether a Freeze Count is registered for a freezing event In the Figure 7 2 the green squares represent observations Freeze Count is recorded only when there is no motion during the observation duration In Figure 7 2 the highlighted green squares are Freeze Counts therefore four Freeze Counts are recorded for the session Note that the frequency of Freeze Counts is greater than the number of Freezing Episodes recorded using the linear method This
33. e unavailable DEO FEAR CONDITIONING JLU FLAN CUINDL LI LUIN 11 NU CHAPTER 9 Recording the Session To begin an experiment load all the animals into the chambers close the doors and click the Record button The Motion Index will record movement and the stimuli will be presented in an automated fashion The experiment is complete when the Motion Index recorder reaches the end of the timeline At the completion of the session the video will stop recording To view the video and motion index data from one chamber click on the data window of the desired chamber Chamber 1 in Figure 9 1 To view the video from all four chambers simultaneously click the Cameras 1 vs 4 tab button located underneath the video display A single click toggles between 1 and 4 chamber views Figure 9 1 The Video Freeze Run Time Screen VFreeze C Documents and Settings VFreezer My Documents VF031405 135515 2 File Experiment Help mia Box 1 Animal Animal 1 Box 2 Animal Animal 2 Group Group Freeze Count 5 Pct Freeze Freeze Count 111 Pct Freeze Cumulative Cumulative Avg Motion jpg Dietantaneous Avg Motion g Instantaneous Box 3 Animal Animal 3 Box 4 Animal Animal 4 Group Group Freeze Count 101 Fet Freeze Freeze Count 111 Pct Freeze Cumulative Cumulative Avg Motion 6 Instantaneous Avg Motion Instantaneous 4 Record Cameras Motion Threshold 00 00 15 000 00 00 30 000 00 00 45 000 00 01 15 000
34. et Working Directorv Protocol Setup Reports O Exit 00 03 30 0 Component Summar Batch Cor ent Summar WA coubnfet gt Auto Scale ay 960 800 WA pocnweyje 640 480 Motion Index 320 160 pseKjob G pocnwenye E Eee T C2 WA peceut TA wa bicmi ez 7 Wa woreic Bans TW em WA pocmueure OG Ela Open Microsoft Excel to view the component summary report Go to Data Get External Data Import Text File choose the csv file to view and select tab delimited transfer Save the component summary report see Table 11 1 as an Excel Workbook xls file i N l Table 11 1 Component Analysis Results csv file in Excel Date 6 14 2008 11 46 Run Time 1800 seconds Protocol Identifiers Experiment CS CS Trial 1 Video Saved Yes Sample Rate fps 30 p fps Method of Motion Threshold au 20 Observation and Detection Method Linear Thresholds Min Freeze Duration 15 frames 00 00 05 seconds Number of Boxes 1 Component Details Component Name CS1 CS2 CSminus1 CSminus2 CS3 CSminus3 Box Animal 1 Animal 1 1 Animal 1 1 Animal 1 1 Animal 1 Start Time Duration Frames Duration sec Start Frame From the 00 30 0 30 900 900 cmp 02 00 0 150 3600 4500 analysis 03 30 0 30 6300 900 file 05 00 0 30 11 10 0 30 07 00 0 30 9000 900 20100 900 12600 900 Dependent Variables
35. final 2 raw 30 min discrimination test 1 txt E Subject 2 final 2 txt 30 min discrimination test 1 wmy Subject 2 final 2 wmv Amygdala 1 wmv A Subject 2 raw My Documents amygdala Phase 1 Trial 1 raw E Subject 2 txt E Amygdala Phase 1 Trial 1 txt Subject 2 wmwv DI Amygdala Phase 1 Trial Lwmv E test1 csv 200000 ED co DI Amygdala Test 1 wmv 2 test raw 8 comparison of CS and novel cmp E test txt 5 e CS CS Comparison cmp F030105_104852 Context 1 wmv 4 o Single Trial US 1 raw E VFO30105 105018 Context 1 raw D My Network Places My Computer 63 E 9 63 E lt Line File name CS CS Comparison cmp Y Files oftype Cancel if To view a summary report of the component analysis go to File Reports Component Summary then type in a file name to save the report as a csv file see Figure 11 4 The csv file is a tab delimited data file that can be read by any word processing or spreadsheet program Figure 11 4 Generating and Saving a Component Summary Report csv file VFreeze C DOCUMENTS AND SETTINGS VFREEZER MY DOCUMENTS SUBJECT 2 FINAL 2 e EN Experiment View Component Help Open Report Component Definition gas ge Abe owws 2605196 CEA Component Na Start Time Duration e i C isa e csi 00 00 30 0 30 01 bigcse cs2 00 02 00 0 150 000 KAMERA HIJS Dane carca cowboneoupebor Export S
36. he Run Time Screen During Calibration gt VFreeze C Documents and Settings VFreezer My Documents VF031505 101635 File Experiment Help Animal Animal 1 Box 2 Animal Animal 2 Group Amygdala Group Amygdala Freeze Count o Pct Freeze Freeze Count p Pct Freeze Cumulative Cumulative Avg Motion o Instantaneous Avg Motion A Instantaneous Box 3 Animal Animal 3 Box 4 Animal Animal 4 Group Control Group Control Freeze Count oj Pct Freeze Freeze Count o Pct Freeze Cumulative Cumulative Avg Motion 0 Instantaneous Avg Motion B Instantaneous Record Cameras D Les n o c 2 ED 00 00 15 000 00 00 30 000 00 00 45 000 00 01 0 000 00 01 15 000 00 01 30 000 00 01 45 000 Select one of the four chambers that appear on the main run time screen by clicking on the data window e g Box 1 Figure 8 1 for the desired chamber Adjust the slider on the right side of the Motion Index graph to move the Motion Threshold the red line into view and then press Calibrate Repeat with each chamber Ensure that all chambers have been appropriately calibrated by verifying that the green motion index line the green line is below the Motion Threshold the red line Once all chambers have been calibrated click the Lock box doing so will enable the Record button to be used to begin the experiment see Recording the Session Once the Lock button has been selected the Calibrate option will becom
37. ile The batch component summary report will contain all of the dependent variables included in the component summary Table 11 2 includes component data for two subjects Rat 3 amp 4 in chambers 1 amp 2 Table 11 2 Batch Component Summary Report Date Motion Threshold Detection Method Min Freeze Duration Component Details Component Name CS1 CS2 CSminus1 CSminus2 CS3 CSminus3 Experiment VF030205 VF030205 VF030205 VF030205 VF030205 VF030205 VF030205 VF030205 VF030205 VF030205 VF030205 VF030205 6 14 2005 13 04 au 20 Linear f 1 Start Time Duration s 00 30 00 30 02 00 0 150 03 30 00 30 05 00 0 30 11100 30 07 00 0 30 Trial Box Animal Group Rat 3 1 05 02 1 Rat 3 1 05 02 1 Rat 3 1 05 02 1 Rat 3 1 05 02 1 Rat 3 1 0 5 02 er Rat 3 1 05 02 1 Rat 4 2 05 02 1 Rat 4 2 05 02 1 Rat 4 2 05 02 1 Rat 4 2 05 02 1 Rat 4 2 05 02 1 Rat 4 2 05 02 1 SD SD SD SD SD SD SD SD SD SD SD SD ComptName Frz Cnt TimeFrz Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Col CS2 Col Cos CS3 CS 3 CS1 CS2 CS 1 CS 2 CS3 CS 3 2 0 167 59 67 567 6 27 667 0 0 0 0 0 0 9 L ad 62 84 2 9 As 0 0 0 0 0 0 STmFrz 0 1 22 5 CompTmFrz 0 6 45 92 2 0 0 0 4 4 56 1 84 3 APPENDIX A Driver and Software Installation If the computer being used with the Video Fear Conditioning System was purchased as part of the system from MED Associ
38. in 39 Video Capturing System Calibration ss 39 Chapter D AM Rene Re Reese RR 40 Recording the SeSSiON a a EE ei EPUM ot de 40 Chapter 10 PRE RER RER 41 Data Anal V SIS est a a te Eis aa a da en 41 ivpesor Data ENS ee ao ea eee nee na 41 Analyzing raw data in Video Freeze rire 42 Exporting Data to a Spreadsheet ss 45 Chapter AMAR 46 Komponent ANAIS S a a aa aaa 46 Component SUMMARY ARS DORE 2 so ds ae 47 Batch Component Summary Report enema nnne hn n enn 50 Appena A LF een sea son a 52 Driver and Software Installation ss 52 ADDED a ananas menaces 54 Door Hinge Ad SCORE EE d PEE 54 Appena C cocus V WENEEEREINNEPEERNII TIN id 56 Camera Filter Care and Handling Instructions 56 Appena Dir 57 Installing Contextual Inserts eene nnnm nnnm nnn 57 ATE gg EE E 57 JMOL FOOL INGEN arri ro iero 57 elg oM Rc RN DR Te 58 MED ASSOCIATES INC VIDEO FEAR CONDITIONING CHAPTER 1 Introduction The Video Fear Conditioning System VFC and the Video Freeze Software represent the latest technological advancements in behavioral research This innovative system allows the researcher to easily construct customized experimental protocols and obtain a quantitative measurement of conditioned immobility or freezing The section on Protocol Setup outlines the steps involved in designing the stimulus conditions for the experiment and the section titled Starting an Experimental
39. k OK to continue These selections may be changed later by clicking Stimulus on the Protocol Setup screen Figure 5 2 Stimulus Selection with User Defined Stimuli Names Stimulus Definition Total Time s 120 Select active stimulus Tone _JStim Light Stimulator Not Used Not Used Not Used Not Used rc L Wh ite L ight INIR Light Now to begin adding stimuli to the protocol click Add and the Add Item screen will appear This function is used to define the stimulus durations and intensities and determines the onset and offset times for each stimulus Use the pull down menu to select the desired stimulus and then enter the Start Time in seconds and the Duration in seconds Click OK to add this item to the protocol A representation of this item will appear on the Protocol Setup screen An example is shown below Figure 5 3 Add Item Add Item Stimulus Start Time s U Duration s U ces MED ASSOCIATES INC VIDEO FEAR CONDITIONING Click Add again to add another stimulus to the protocol or right click on the stimulus representation shown on the Protocol Setup screen and click Copy Then right click near the desired start time for the item and select Paste A representation of the new stimulus will appear Click on a stimulus representation the red or green rectangle to modify the properties of the stimulus Properties may be modified either by changing the
40. l be represented in the data file for export to Excel see the next section for instructions on exporting Exporting Data to a Spreadsheet Export the txt data file to a desired location on the desktop computer by selecting File Export Figure 10 7 Exporting a Data File 79 Video Freeze Experiment Help Animal Animal 3 ved i Selected Box Cursors Set Working Directory SEIT Dn C Box 1 Box Times Range Delta X1 X2 L ii Protocol Setup miu o 00 00 00 33 X1 00 00 50 0 00 00 20 33 Mi X2 00 01 10 33 Reanalysis Parameters Freezing Measures 20 Method Movement Threshold Recalc Avg Movement 9 2 Freeze Count e Linear Min Freeze Duration f 15 00 00 00 500 Time Freezing 14 833 Discrete Percent Freeze 74 0 39 862 00 00 45 782 00 00 51 702 00 00 57 622 00 01 3 542 00 01 9 462 00 01 15 382 00 01 21 302 00 01 3 Once the file has been exported then use Microsoft Excel to open a blank spreadsheet and select Data Get External Data Import Text File Choose the desired file and the import wizard will appear Choose tab delimited transfer Figure 10 8 Importing a Data File Into Microsoft Excel Fi Be Edt View Insert Format Took D 1D Gad E 4 Ba H sot A1 Y F3 i B ieu Text Import Wizard Step 1 of 3 The Text Wizard has determined that your data is Delimited Tf this is correct choose Next or choose the data type that best describes your data
41. le files at once press the button Figure 11 6 Select the raw File or Files Select file s to report on Lookin GJ My Documents m 6 My Recent Documents Desktop My Documents My Computer My Network Places ck Ev my Music My Pictures Emy videos E 30 min discrim Test 1 raw E 30 min discrimination test 1 raw E Amygdala Phase 1 Trial 1 raw E Single Trial US 1 raw Biss 2 30 min 2 raw Subject 2 final 2 raw B Subject 2 raw E test raw a VFO30105_105018 Context 1 raw VFO30105 110900 Context 2 raw E VFO30105_ 112300 Context 3 raw s VF030105 160803 raw s VF030205 095511 raw E VFO30205_ 103900 Tone 1 raw A VFO30205_ 110400 Tone 2 raw E FO30205_ 112000 Tone 3 raw tj VFO30205 113500 tone 4 0 raw B VFO30205 113500 tone 4 raw El VFO30205_114856 raw fe VFO30205 134528 Trail 1 raw E VFO30205 134528 Trail 2 raw B VFO30305 132728 Context 1 raw E VFO30305_ 134528 Rat Context 2 raw E VFO30305_ 150028 Context Rat 3 raw tA VFO30205 144828 Rat Condition 3 raw E IE B VFO30105 113500 Context 4 raw E VFO30405_090459 raw File name Files of type Subject 2 final 2 raw Raw datafiles raw Open as read only Ctrl key while using the left mouse Upon selecting the raw data files the batch component analysis will be saved to the location specified for the output f
42. ment Motion Threshold See Motion Threshold Section below Sample Rate The sample rate refers to the number of video frames scanned per second This value is fixed at 30 fps Method See Chapter 7 Save Select Save to save the experimental configuration exp for later use and choose Load to use a previously saved experiment file Ok To start the experiment Cancel To cancel and exit Threshold Values The subject s behavior is quantified as an ndex of motion All movement within the conditioning chamber is registered by the software as a change in video pixel composition over time Therefore robust and fast movements will be recorded as a large relative changes in video pixel composition and small refined and slow movements will be registered as somewhat smaller relative changes in pixel composition The index of motion has a range of 0 76 800 and during an experiment movement is represented as a graph motion index vs time in a non cumulative manner Motion Threshold The Motion Threshold au refers to an arbitrary limit above which all behavior will register as movement i e not freezing in the index of motion When movement falls below this threshold this behavior or lack of behavior will be counted as freezing A Motion Threshold of 10 20 au is recommended NOTE The Motion Threshold can be modified after the experiment is complete during data analysis The Method of Observation can also be changed in d
43. n the focus ring shown in Figure 4 1 Once proper image clarity is achieved tighten the setscrew on the focus ring It is recommended that the lens aperture be locked in the maximal open position using the set screw on the aperture ring Figure 4 1 Make sure that the white dot on the aperture ring is lined up with the number 1 4 on the lens barrel Figure 4 2 This is the maximally open aperture position Figure 4 1 Focus and Aperture Rings on Video Lens Setscrews 16 8 4 1 4 Focus Ring PEAR ci FAR gt Figure 4 2 Aperture Setting Proper Aperture Setting C 16 8 4 1 4 DEAR FAR gt 22 SSOCTATES Il Calibration Procedure 1 Ensure that the NIR light source is turned on in each box It is important to note that the cameras in the MED VFC system detect only NIR light and therefore there will be no image on the screen if the NIR light source is off Select File Chamber Configuration The following screen will appear Figure 4 3 Configuration Manager Configuration Manager Camera Id Box Workstation 2 Box 2 Workstation 3 Box 3 White Workstation 4 Box 4 Add Edit Delete The order that the Camera Ids are displayed in on the Configuration Manager screen can be changed by left clicking on the camera to move then clicking on the up or down arrow on the right side of the display Figure 4 3 Camera Ids may be removed from this list by highlighting them and selecting Delete
44. nnection Panel For example if there are multiple chambers the VFC 100 card labeled 1 should be connected to the VFC 716 in Chamber 1 and so on NOTE All cables going in to the Sound Attenuating Cubicle should be run through the opening on the right side VFC 716 OUTPUTS INPUTS 1 see 1 bee 2 eee ees 3 bee 3 bee dii 6 Ga ar 5 ses VFC 716 6 see Passive 7 ees Connection SG 6080D see Panel Cabinet h lt er Gh CET vcio Er us All cables running into the Sound Attenuating Cubicle SAC should be run through the grommet plate on the right side of the SAC 4 Using an SG 219G 10 DB 9 cable connect the Grid Output connector on the back of the ENV 414S Aversive Stimulator to the ENV 005 QD grid floor on the rear of the ENV 008 FC Conditioning Chamber inside the Sound Attenuating Cubicle SG 219G 10 Cable Grid Floor Harness NOT FOR HUMAN USE 50 kO DUMMY AM Q MED o GRID OUTPUT Control LOAD Vour B ame EET LN CAUTION ew 110 Volts HIGH VOLTAGE Fuse 0 5 Amp SB TTL Switch Closure Back of ENV 414S Aversive Stimulator All cables running into the Sound Attenuating Cubicle SAC should be run through the grommet plate on the right side of the SAC 12 MED ASSOCIATES INC VIDEO FEAR CONDITIONING 5 Using an SG 216A 10 3 pin Molex cable connect the MED Control connection on the back of the ENV 414S Aversive Stimulator to the OUTPUT 2 connection on
45. onnected Mot Connected NIR House Light White House Light ANL926 DIG 704 I O DIG 704 I O DIG 704 I O DIG 704 I O DIG 704 I O DIG 704 I O DIG 704 I O DIG 704 I O 20 MED ASSOCIATES INC VIDEO FEAR CONDITIONING The Stimulus Names screen Figure 3 2 is shown with the default titles The Name column is the only column that may be changed the Id and Type columns may not be changed To change the Name of a stimulus click on the desired name and enter the new name When all the names are correct select Ok Now select File Camera Identities The following screen will appear Figure 3 3 Camera Setup Camera Setup Select the camera to edit Camera 1 This feature mav be used to both name the cameras and identifv which camera is in which chamber The camera name can be changed by clicking on the current camera name on the left side of the screen and entering a new name The image on the right side of the screen corresponds to the selected camera When the camera names have been updated select Exit 21 MED ASSOCIATES INC VIDEO FEAR CONDITIONING CHAPTER 4 Camera Calibration Adjusting Camera Focus MED Associates adjusts the focus and aperture settings on the camera prior to shipping Video Fear Conditioning systems However if focus adjustments are made the camera settings Brightness Gain Shutter will need to be adjusted using procedure below To adjust the camera focus loosen the setscrew o
46. ons inter trial intervals inter stimulus intervals session durations and number of trials per session These variables are defined when constructing a protocol see Protocol Setup Dependent Variables Video Freeze Software is an important advancement in behavioral testing technology because it provides a reliable automated means of monitoring the effects of fear conditioning in rodent species The consequence of fear conditioning in rodents is freezing defined as no movement other than breathing therefore freezing is the primary dependent variable Freezing is defined using two thresholds see Threshold Values and freezing is represented in the data file in several different ways see Analyzing raw Data in Video Freeze For instance data can reflect the total time the subject spends motionless during the session the percent of time motionless and the number of freezing episodes or occurrences Furthermore the software also generates an average ndex of motion to provide researchers with data on overall activity Hardware Guide Figure 1 1 ENV 022MD Sound Attenuating Cubicle with Chamber ENV414S Aversive Stimulator VFC 716 Connection Panel NIR 100 Light Controller amp NIR 100R Light Panel MED ASSOCIATES INC VIDEO FEAR CONDITIONING Figure 1 2 ENV 008 FC Conditioning Chamber with VFC 716 Passive Connection Panel VFC 716 a OUTPUTS INPUTS House Light y 1 ooo ENV 414S A ive Stimulat versive S
47. rayscale Histogram Display Figure 4 7 Figure 4 7 Narrow Peak on the Grayscale Histogram Display Calibrate Narrow Grayscale Band Average Intensity Increase Brightness standard Deviation AG EN pem LI 1 150 200 250 I I 1000 1500 10 Move the Shutter slider to the right An image should start to form in the Video Display Window and the Grayscale Histogram should start to spread out Continue moving the Shutter slider to the right until the right most edge of the histogram curve is at the right most edge of the Grayscale Histogram Display Figure 4 8 Spreading the Grayscale Histogram to the Right by Increasing Shutter Calibrate Grayscale Histogram Spread to Right Edge Average Intensity Standard Deviation Brightness 2 a 773 3D Gain l Z 50 100 Increase Shutter I 300 11 Now decrease the brightness by moving the slider to the left until the left most edge of the histogram curve is at the left edge of the Grayscale Histogram Display Notice that the entire Grayscale Histogram has moved leftward and there is a gap between the right edge of the histogram and the right edge of the Grayscale Histogram Display Figure 4 9 Shifting the Grayscale Histogram to the Left by Decreasing Brightness Calibrate Grayscale Histogram Shifted to Left Edge Gap on the Right Average Intensity Standard Deviation Decrease Brightness ina Gain 1
48. reezing Measures Avg Movement 70 4 Freeze Count 7 Time Freezing 40 233 33 5 Percent Freeze Movement Threshold 20 Recalc Min Freeze Duration f Delta X1 X2 00 00 00 000 Range X1 00 00 00 000 X2 00 00 00 000 Freezing Measures 70 4 7 40 233 33 5 Avg Movement 15 00 00 00 500 Freeze Count Time Freezing Percent Freeze 00 01 9 160 00 01 13 590 00 01 18 020 File Experiment Help Animal Animal 3 Group Control Selected Box C Box1 Box2 e Box3 Box4 Range Delta X1 X2 X1 00 00 53 366 00 00 10 873 X2 00 01 04 240 Time 00 00 00 33 Reanalysis Parameters Movement Threshold 20 15 00 00 00 500 Time Freezing 10 867 Recalc Min Freeze Duration f The figure below displays the Range cursors Note that the Freezing Measures reflect only the data that exist between the two vertical lines on the Motion Index Graph Figure 10 6 Positioning the Range Cursors 7 Video Freeze Fle Experiment Help Animal Animal 3 Group Control Selected Box Cursors Boxi Box2 Time Range Delta X1 X2 00 00 00 33 X1 00 00 50 0 00 00 20 33 X2 00 01 10 33 Reanalysis Parameters Freezing Measures 20 Method Movement Threshold Recalc Avg Movement 9 2 e Linear Min Freeze Duration f 15 00 00 00 500 Freeze Count 1 Time Freezing 14 833 Discrete Percent Freeze 74 0 NOTE If the range cursors are selected only the data between the two cursors wil
49. save a csv file that contains multiple experimental session data To generate a batch component summary report click the first button marked and select a cmp file Then name the batch report using the second button Select a Method of Observation Linear or Discrete and define the threshold values These setting will be applied to all of the raw data included in the batch summary report see Table 11 1 Figure 11 5 Generating a Batch Summary Report Using Multiple raw Data Files VFreeze C DOCUMENTS AND SETTINGS VFREEZER MY DOCUMENTS SUB JECT 2 FINAL 2 Experiment View Component Help Open Report Component Definition Export Component Na Start Time Duration CS1 00 00 30 0 30 000 CS2 00 02 00 0 150 000 CSminusi 00 03 30 0 30 000 Set Working Directorv Protocol Setup CSminus2 00 05 00 0 30 000 00 11 10 0 30 000 Component Summary 00 07 00 0 30 000 Exit Batch Component Summa Co mpo ne nt Summary Report a e w m avec Component File CS CS Comparison cmp n Output File Batch Report csv 640 puo e Linear Descrete 320 160 i Motion Threshold 20 Min Freeze Duration f 15 0 000 00 03 45 000 00 07 30 000 00 11 15 000 00 15 0 000 00 18 45 000 00 22 30 000 00 26 15 000 00 30 zx d o E 2 B fa z OK Cancel Select OK and then choose the raw file or files to which the cmp analysis will be applied To select multip
50. sion time represented in the immediately available motion index display Click Auto Scale to return to a view of the entire session Figure 10 4 Adjusting the 7 Video Freeze File Experiment Help i 00 00 51 440 Range Cursors To obtain dependent measures from a specified interval of time within the session use the Range Cursors Select the Range Cursors with a right click on the motion index graph see Figure 10 5 and then choose the Move Cursors Move Cursors to Center option Adjust the positioning and length of the specified interval by moving the vertical blue lines with a left click of the mouse Drag the blue lines to the desired position in the Motion Index Graph and then press Recalc Adjusting the position of the Range Cursors will alter the data listed under the Cursors display on the data analysis screen Figure 10 5 Selecting the File Experiment Help Animal Animal 3 Group Control Selected Box C Box1 Box2 Time e Box3 Box4 Sean TKE RSE Reanalysis Parameters Method Movement Threshold e Linear Min Freeze Duration f C Discre te Bka Auto Scale 00 00 55 Blue Timeline Animal Animal 3 Group Control Selected Box C Box1 Box2 Cursors Time 00 00 00 33 Reanalysis Parameters Method e Linear Discrete 870 00 01 0 300 00 01 4 730 Range Cursors Range Delta X1 X2 X1 00 00 00 000 00 00 00 000 X2 00 00 00 000 20 Recalc 15 00 00 00 500 F
51. timulator Tan 3 ooo 4 ooo White Light Control NIR Light Control Speaker Connection o 28 Volts Serial Port Cable from Interface Figure 1 4 ENV 414S Stand Alone Aversive Stimulator Scrambler and ENV 005 QD Quick Disconnect Harness US Pat No 6412441 BI Canadian Pat No 2 368 344 UK Pat No 1226750 Australian Patent 772111 France 1 226 750 Germany 602 05 143 6 Italy 1 226 750 The Netherlands 1 226 750 European Pat No 1226750 Other Foreign Patents Pending MED ASSOCIATES INC VIDEO FEAR CONDITIONING Figure 1 5 Fire Wire Video Camera with CAB FIRE I 3M 6P RA Cable Figure 1 7 SG 6080D Cabinet with DIG 700F PCI Interface and VFC 100 Output Modules MED ASSOCIATES INC VIDEO FEAR CONDITIONING Figure 1 8 DIG 704PCI Interface Card Back of Computer Figure 1 9 NIR 100 Light Controller Front Panel med ASSOCIES ING White Light NIR Light Remote em ON Remote em ON Calibrate Calibrate Power Light ON Light ON e NC NIR 100 Figure 1 10 NIR 100 Light Controller Back Panel 15 to 28V no no onggo a NIR Light White Light To Light Panel Control Input Control Input Figure 1 11 NIR 100R Light Panel Mounted on top of the Cubicle Table 1 1 Part Number VFC 022MD ENV 008 FC SG 6080D DIG 700F VFC 100 DIG 704PCI ENV 4145 ENV 005 QD VFC 716 ENV 227M VFC CAMERA 4 3MM NIR 100 NIR 100R FIN ENV 025F
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