DOC-040 - Med Associates

DOC-040 - Med Associates
instrumentation and software for research
STARTLE REFLEX
SOF-825
USER’S MANUAL
DOC-040
Rev. 6.3
Copyright ©2016
All Rights Reserved
Med Associates Inc.
P.O. Box 319
St. Albans, Vermont 05478
Phone: 802.527.2343
Fax: 802.527.5095
www.med-associates.com
notes
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Table of Contents
Chapter 1 | Introduction ................................................................................................1
Chapter 2 | Hardware and cable overview ................................................................ 2
Hardware Guide............................................................................................................................. 2
Cable Guide .................................................................................................................................... 4
Chapter 3 | System Setup ..............................................................................................6
Software Installation..................................................................................................................... 6
Hardware Installation ................................................................................................................... 6
Chapter 4 | Calibration ................................................................................................. 13
Audio Calibration ......................................................................................................................... 13
Calibrating Chamber 1 ................................................................................................................ 13
White Noise Calibration .............................................................................................................. 17
Pure Tone Calibration ................................................................................................................. 17
Background Noise Calibration ................................................................................................... 18
Calibrating Additional Chambers ............................................................................................... 19
Input Calibration.......................................................................................................................... 20
Chapter 5 | Running Experiments ............................................................................. 25
Sample Experiments ................................................................................................................... 25
Glossary of Important Terms .................................................................................................... 25
Prepulse Inhibition (PPI) ............................................................................................................ 26
PPI Timing .................................................................................................................................... 27
Prepulse Inhibition (PPI) Sample Experiment ......................................................................... 27
Special Options Utility ................................................................................................................. 34
Fear-Potentiated Startle (FPS) .................................................................................................. 36
FPS Timing ................................................................................................................................... 37
Fear-Potentiated Startle (FPS) Training Sample Experiment................................................ 38
Fear-Potentiated Startle (FPS) Testing Sample Experiment ................................................. 41
Running PPI or FPS Experiments .............................................................................................. 46
Saving Data from PPI or FPS Experiments .............................................................................. 46
References.................................................................................................................................... 46
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Chapter 6 | Menu Selections ....................................................................................... 47
File Menu Options ....................................................................................................................... 47
Hardware Menu Options ............................................................................................................ 47
Config Menu Options .................................................................................................................. 51
Data Menu Options ..................................................................................................................... 52
Run Menu Options ...................................................................................................................... 54
Graph Menu Options ................................................................................................................... 55
Window Menu Options ............................................................................................................... 56
About Menu Selection ................................................................................................................. 56
Chapter 7 | Saving And Exporting Data.................................................................... 57
Computed Values ........................................................................................................................ 57
Raw Data ...................................................................................................................................... 57
Statistical Data............................................................................................................................. 58
Opening A Startle Database With Excel ................................................................................... 60
Importing a Text Data File Into Excel ...................................................................................... 65
File Formats ................................................................................................................................. 67
Chapter 8 | Alternate Calibrate Input Utility........................................................... 68
Chapter 9 | Startle Units Explained ........................................................................... 70
Chapter 10 | Contact Information ............................................................................. 72
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CHAPTER 1 | INTRODUCTION
The startle reflex is a motor response to an intense and unexpected stimulus. The anatomy of
the acoustic startle reflex is well-characterized and is often studied in humans as well as rodents
to measure sensorimotor processing, emotional state, and/or attention. The amplitude of a
study subject’s response (usually a “jump”) is quantified. This “jumping” reflex response can be
altered in different strains of animals, by pre-pulse inhibition of startle (PPI), and in fearpotentiated startle (FPS). This Startle Reflex system allows users to obtain accurate
measurement of the acoustic startle reflex in rodents.
The Startle Reflex software package is designed for use with Med Associates modules to
produce stimuli, collect response data, plot data to the screen, and perform waveform analysis.
Please thoroughly read this manual prior to setup in order to gain an understanding of the
Startle Reflex system.
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CHAPTER 2 | HARDWARE AND CABLE OVERVIEW
Hardwar e Gui de
Figure 2-1 - The Startle Reflex System
Figure 2-2 - Startle Reflex Cabinet with ANL-729, ANL-925E and ANL-925D
Figure 2-3 - Sound-Attenuating Cubicle (SAC)
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Figure 2-4 - 255A Stimulus Connection Panel with Platform Table & Speakers
Figure 2-5 - PHM-250B Startle Load Cell Amplifier Front and Back Panel
Figure 2-6 - ENV-414S Constant Current AversiveENV-414S
Stimulator Front and Back Panel
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Figure 2-7 - Startle Reflex System Assembled
Cable G uide
Table 2-1 - Startle Reflex Cable Guide
Part Number
Image
SG-244 68-pin NI PCI-6023E Interface Cable
SG-USB-6 USB Cable
SG-210CB-25 Serial Port Cable
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SG-216A-10 3-Pin Molex Cable
SG-219G-10 DB-9 Shock Cable
SG-250A PHM-250B to PHM-255A Cable
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CHAPTER 3 | SYSTEM SETUP
NOTE: Always turn off the power prior to performing work on the computer or interface
cabinets. Serious damage or personal injury may occur if this precaution is not taken.
Software I nstall ation
If the computer was purchased as part of Startle Reflex system from Med Associates, the driver
and software installation and the DIG-744/DIG-744E PCI card installation was completed at the
factory. If the computer was not purchased from Med Associates or was not a part of the
system, follow the instructions below to install the drivers, software and DIG-744/DIG-744E PCI
card.
Before beginning the software 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 software installation process.
Insert the Startle Reflex CD into the CD drive and at the welcome screen click Install. If the CD
does not auto-start, navigate to the CD drive in Windows Explorer and double click the
“autorun.exe” file and click Install at the welcome screen. Successful installation of each
component will be indicated by a green check mark, a red X will indicate an unsuccessful
installation. When prompted enter the user name, company name and software installation
password. When the installation completes, click Finish.
Once the software and driver installation is complete, turn off the computer and install the
National Instruments PCI-6023E (DIG-744) or PCIe-6320 (DIG-744E) card, following the
instructions included with the computer for installing PCI devices. After the card is installed,
reboot the computer.
Hardwar e Installati on
1.
Place the PHM-255A Stimulus Connection Panel with Platform Table and Speakers, and the
PHM-250B inside the Sound-Attenuating Cubicle (SAC) as shown in Figure 3-1.
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Figure 3-1 - Startle Platform in SAC
2.
Loosen the two Philips screws on the grommet plate (right side of SAC) shown in Figure 3-2
and remove the top half. All cables should be routed into the SAC through this cable port.
Figure 3-2 - Cable Port
3. Using the SG-244 68-pin Interface Cable, connect the DIG-744 PCI card (back of
computer) to the ANALOG DATA port on the ANL-729 card (Interface Cabinet). See
Figure 3-3.
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Figure 3-3 - Connect DIG-744 PCI Card to ANL-729 Card
4. Using the SG-USB-6 Cable, connect any available USB port on the computer to the USB
port on the ANL-729 card (Interface Cabinet) shown in Figure 3-4.
Figure 3-4 - Connect USB Port to ANL-729 Card
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5. Using the SG-210CB-25 Cable, connect the CHAMBER CABLE port on the PHM-255A
Stimulus Connection Panel to the Chamber Cable port on the ANL-925D card (Interface
Cabinet). The leftmost ANL-925D card corresponds to Chamber 1; the second card
corresponds to Chamber 2, etc (see Figure 3-5).
Figure 3-5 - PHM-255A Startle Platform to ANL-925D Card
6.
(Optional) Using the SG-216A-10 Cable, connect the AUX3 port on the PHM-255A Startle
Connection Panel to the MED CONTROL port on the ENV-414S Aversive Stimulator shown
in Figure 3-6.
Figure 3-6 - PHM-255A Startle Platform to ENV-414S Aversive Stimulator
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Connect the Startle Platform Cable to the LOAD CELL port on the PHM-250B shown in
Figure 3-7.
Figure 3-7 - Startle Platform to PHM-250B
8.
Using the SG-250A Cable, connect the POWER/SIGNAL OUT port on the PHM-250B to the
PLATFORM port on the PHM-255A Stimulus Control Panel shown in Figure 3-8.
Figure 3-8 - PHM-255A Startle Platform to PHM-250B
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(Optional) Connect the Fluorescent Stimulus Light PHM-258L to the Fluorescent Lamp
Controller PHM-258 shown in Figure 3-9.
Connect Fluorescent Lamp Controller PHM-258 to an available AUX port on the PHM-255A
Stimulus Control Panel shown in Figure 3-10.
Figure 3-9 - Connect PHM-258L Lamp to PHM-258 Lamp Controller
Figure 3-10 - Connect PHM-258 Lamp Controller to PHM-255A Stimulus Control Panel
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10. (Optional) Using the SG-219G-10 Cable, connect the GRID OUTPUT port on the ENV-414S
Aversive Stimulator to the Grid Harness shown in Figure 3-11.
Figure 3-11 - ENV-414S Grid Output to Grid Harness
11. Connect the fan cable (inside the SAC) to the FAN port on the PHM-255A shown in Figure
3-12.
Figure 3-12 - Connect Fan to PHM-255A
12. It is now safe to apply power to the Startle Reflex Cabinet, ENV-414S (optional), PHM-258L
(optional), and computer.
13. Replace the top half of the grommet plate and tighten the Philips screws.
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CHAPTER 4 | CALIBRATION
After the Startle Reflex system has been properly installed (see Chapters 2 & 3), it is important
to test the system to ensure that it is functioning properly. A complete calibration procedure is
performed prior to shipping; however, it is necessary to perform the audio and load cell
calibrations for each new protocol and/or set of animals.
Audio C alibr ation
Due to the fact that each speaker has a slightly different frequency response, and that different
frequencies are produced at naturally different volumes, it is important to perform the audio
calibration for each chamber. By using the Calibrate Audio utility and the ANL-929A-PC USB
Microphone Package, each speaker's frequency response can be tested and the results can be
used to correctly present the desired frequencies at the specified volume.
This system includes all components necessary for calibrating the speaker(s) to generate
reproducible startle stimuli. For more detailed information regarding the installation and use of
the ANL-929A-PC USB Microphone Package, refer to the ANL-929A-PC User’s Manual, DOC-089.
Calibrati ng Chamber 1
Before performing Audio Calibration, determine the sound parameters of the testing protocol.
This includes the decibel (dB) levels for the white noise, background noise, and the dB level and
frequency (Hz) for the pure tone(s).
Recommended audio calibration method:
1. Install the ANL-929A-PC software and microphone according to the ANL-929A-PC
User’s Manual, DOC-089.
2.
Place the ANL-929A-PC microphone inside the desired animal holder, using the
microphone calibrator that came with your animal holder(s).
Examples of
microphone calibrator placement are shown inFigure 4-1.
Figure 4-1 - Microphone calibrators
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3. Place the animal holder with the microphone calibrator on the startle platform
inside Chamber 1, being sure to orient the holder so that the holes are facing
towards the speaker at the back of the chamber. Align the right hand edge of the
startle platform with the right hand edge of the base of the PHM-255A (red arrows,
Figure 4-2). Use the platform adjustment knob (green arrow, Figure 4-2) to make this
adjustment.
Figure 4-2 - Startle Platform Aligned With Base Plate
NOTE: Take careful note of the microphone and platform placement in Chamber 1. Be sure
to orient the startle platform and microphone in exactly the same position in each chamber
being calibrated.
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4. Set the background noise and acoustic startle dials on top of the PHM-255A to 0 and then
close the doors on the startle chamber.
Figure 4-3 - PHM-255A Acoustic Startle Adjustment Knob
5. Launch the ANL-929A-PC Sound Level Meter software application (Figure 4-4) and
drag it to a position near the bottom of the screen. Keep the application open and
visible for the following steps.
Figure 4-4 - Sound Level Meter Software
6. Launch Startle Reflex and resize the application window so that the ANL-929A-PC
Sound Level Meter software window is also visible. From the Startle Reflex main
menu bar select Hardware > Calibrate Audio. The Calibrate Audio screen (Figure
4-5) will appear.
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Figure 4-5 - Calibrate Audio Screen
Table 4-1 - Calibrate Audio Screen Options
Item
Description
Record Frequency Response
Used to set the frequency offset to adjust for varying
speaker frequency responses.
Adjusted Single Tone Test
Tests a single tone using the offset.
Adjusted Frequency Sweep Test
Used to confirm offsets applied during the Record
Frequency Response test are correct. See Pure Tone
Calibration.
Execute Test
Begins the selected test.
Show Response
Displays the offset table created from running the
Record Frequency Response test.
White Noise On
Locks on the white noise.
White Noise Off
Turns white noise off.
Frequency (Hz)
Sets frequency used for Adjusted Single Tone Test.
Start Frequency (Hz)
Beginning (lowest) frequency being used in a
protocol.
Stop Frequency (Hz)
Ending (highest) frequency being used in a protocol.
Step Size (Hz)
Size by which played frequency will be incremented.
With a Step size of 500Hz there would be 20 steps.
15KHz – 5KHz = 10KHz / 500Hz = 20 steps. Played
frequencies would be 5KHz, 5.5KHz, 6KHz,
6.5KHz…15KHz.
Loudness (dB)
Amplitude (volume) at which frequencies will be
played.
Exit
Exits the Calibrate Audio Screen
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White N oise C alibrati on
7. Set the Loudness (dB) to 80dB and click the White Noise On button. Verify the
Sound Level Meter Software (Figure 4-4) displays approximately 80dB. Repeat these
steps using 110dB. If the value displayed in the Sound Level Meter Software differs
from the set value by a significant amount please contact technical support (see
Contact Information) for detailed information on adjusting the internal trimming
potentiometers.
NOTE: White Noise bursts and pure tones are both produced by the right-hand speaker on
the PHM-255A.
Pure Tone Calibr ation
8. Enter the lowest frequency being used in the protocol in the Start Freq (Hz) box.
Enter the highest frequency in the Stop Freq (Hz) box, the step size in the Step Size
(Hz) box and the median loudness used in the protocol in the Loudness (dB) box.
Refer to Figure 4-5.
9. Select the Record Frequency Response test and press Execute Test. The Record
Frequency Response screen (Figure 4-6) will open.
Figure 4-6 - Record Frequency Response Screen
10. Use the Up/Down arrows to adjust the loudness displayed in the Sound Level Meter
Software to match the Desired Loudness (db). Press Continue when the two values
are as close as possible. Pressing Quit will result in an incomplete calibration.
11. Select Adjusted Frequency Sweep Test and set the Loudness (dB) to the minimum
pure tone dB level used in the protocol.
12. Press Execute Test and confirm the dB level shown in the Sound Level Meter
Software approximates the desired pure tone dB level, see Figure 4-7. Press
Continue to step through each frequency.
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Figure 4-7 - Adjusted Frequency Sweep Test
13. Enter the maximum pure tone dB level used in the protocol in the Loudness (dB)
field. Repeat step 12. When complete click Exit.
14. If any of the values for the minimum or maximum dB levels significantly differ from
the desired level, contact support (see Contact Information) for detailed instructions
regarding adjusting the internal trimming potentiometers.
Background N oise Cali brati on
15. To calibrate Background Noise, click Hardware > Stimulus Test and set the dB level
to 70dB in the Background Level (dB) field on the Stimulus Test screen (Figure 4-8).
16. Click Background Noise Lock On.
17. Verify the dB level using the Sound Level Meter software.
18. Repeat using a Background Level (dB) of 90dB. If the value displayed in the Sound
Level Meter Software differs from the set value by a significant amount please
contact technical support (see Contact Information) for detailed information on
adjusting the internal trimming potentiometers.
NOTE: Background noise is produced by the left-hand speaker on the PHM-255A.
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Figure 4-8 - Stimulus Test Screen
Calibrati ng Additi onal Chambers
1. Chamber 1 is used as a reference when calibrating additional chambers. For this reason it is
important to position and orient the microphone in subsequent chambers precisely as it was
in chamber 1.
2. To match additional chambers to Chamber 1, place the microphone and holder in the
next chamber to be calibrated exactly as it was in Chamber 1 and select Adjusted
Single Tone Test from the Calibrate Audio screen. Enter the median pure tone
frequency that will be used in the Frequency (Hz) field and enter the desired volume
in the Loudness (dB) field.
3. Select Execute Test and the screen shown in Figure 4-9 will appear.
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Figure 4-9 - Adjusted Single Tone Test
4. A tone will now be generated at the desired Frequency (Hz). Using the Sound Level
Meter software, compare the actual dB level being displayed to the set dB level.
5. Repeat steps 1 – 4 for each of the remaining chambers and use the acoustic startle
adjustment knob (right hand knob, Figure 4-3) on the top of the PHM-255A to adjust
the actual dB level until it is equal to the set dB level.
It is important to note that the human ear will incorrectly perceive certain frequencies
to be louder than others, even though a dB meter will show the same loudness level for
both.
Input Calibr ation
Proper calibration of the load cell ensures consistent measurements across multiple chambers.
Startle Reflex software utilizes an analog-to-digital converter that converts the analog voltage
signal from the startle sensor to a digital unit having a value between –2048 and +2048.
Use the following calibration procedure to ensure that each Startle Reflex chamber produces
similar data output.
Note: Some older versions of the hardware require the use of a spinner calibrator. Please
refer to Chapter 9, Alternate Calibrate Input Utility for input calibration using a spinner.
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The input calibration procedure has two components:
•
Adjust Tare (offset) so that the signal amplitude is zero over a range of gain settings.
The Tare should be adjusted while only the animal holder is on the load cell platform.
This ensures a consistent reading of zero is obtained with no applied force.
•
Apply a known weight and adjust the gain so that the signal amplitude is at the same
reference value in all chambers. This procedure ensures that a given amount of force
will result in the same signal in all chambers.
1. From the Hardware Menu, select Calibrate Input. Enter the number of the chamber
to calibrate in the Chamber field and select Weight from the Calibrator Type drop
down menu (Figure 4-10).
Figure 4-10 - Calibrate Input
2. Set the GAIN knob on the front of the PHM-250B amplifier to 0.1 (Figure 4-11).
Figure 4-11 - PHM-250B amplifier
3. If working with mice, switch the RAT/MOUSE toggle switch to MOUSE. If working
with rats, set the toggle switch to RAT.
4. Set the CAL/RUN switch to CAL.
5. Place the animal holder that will be used during the experiment on the load cell
platform.
6. Click Begin (Figure 4-10). The screen shown in Figure 4-12will appear. The signal
from the PHM-250B platform amplifier is displayed as a red line.
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Figure 4-12 - Amplifier Signal at 0 Marker
7. Use a small screwdriver to adjust the TARE potentiometer located on the front of the
PHM-250B amplifier until the signal is as close to zero as possible. Some slight
fluctuations are normal, as the signal from the load cell is affected by environmental
conditions such as air currents and vibrations.
8. Turn the TARE potentiometer slowly clockwise to increase the output amplitude and
move the red line up. Turn the TARE potentiometer slowly counter-clockwise to decrease
the output amplitude.
NOTES: Turn the TARE potentiometer gently and very slowly, as it is possible to turn it too
far, and not be able to adjust the signal any further. If you have turned the potentiometer
too far, you may hear a faint click upon each revolution. If this occurs, slowly turn in the
opposite direction.
If the signal does not respond as the potentiometer is turned, ensure that the same
chamber listed in the Calibrate Input dialog matches the chamber being tested. Trace the
cable from the PHM-250B back to the interface cabinet to validate the chamber number.
Reduce the gain, and turn the potentiometer more slowly.
9. Increase the GAIN knob on the front of the PHM-250B amplifier to 2.0.
10. Adjust the TARE potentiometer until the signal is as close to zero as possible.
11. Increase the GAIN knob on the front of the PHM-250B amplifier to 3.0.
12. Adjust the TARE potentiometer until the signal is as close to zero as possible.
13. Repeat steps 9-10, increasing gain in increments, until the amplifier is tared at the highest
setting desired.
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14. Decrease the GAIN to 1.0. The load cell signal should stay near zero, even while you are
decreasing the gain.
15. To calibrate the platform for mice, place 40 grams of weight inside the animal holder (on
the load cell platform). For rat calibration, use 300 grams.
NOTE: These are typical weights used, but any known weight can be used.
For a long-term experiment that begins with juvenile animals and ends with adult animals,
be sure to calibrate using test weight that is appropriate for an adult animal.
16. Adjust the gain knob on the front of the PHM-250B until the signal is in line with the 240
marker on the vertical scale on the left of the screen, as shown in Figure 4-13. (Vertical
scale units are arbitrary.)
17. Remove calibration weight from the test platform and verify that the signal returns to
zero.
Figure 4-13 - Amplifier Signal at 240 Marker
18. Set the CAL/RUN switch on the PHM-250B to RUN and click DONE.
19. Repeat this procedure for each Startle Platform.
NOTE: It is best to run a few test experiments with animals within an expected startle
response range for your studies before acquiring data. This is to ensure your input
calibration is within the ideal range for your particular experiments, as different individual
animals or groups of animals may have different startle ranges or weights.
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If gain is set too low:
If gain is set too low, the system may report reduced or absent startle response. To fix this, recalibrate using a decreased amount of test weight. Then, adjust gain knob on the PHM-250B
until the signal is in line with the 240 marker on the vertical scale.
Figure 4-14 - Gain too low: Reduced or Absent Startle
If gain is set too high:
If gain is set too high, you may observe “clipping”, which occurs when the amplifier is
attempting to produce a response that exceeds its maximum voltage capability. To fix this, recalibrate using increased test weight.
Figure 4-15 - Gain too high: Clipping
NOTE: If you have re-calibrated any one chamber with a different amount of test weight,
you must re-calibrate all the other chambers with the same amount of test weight.
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CHAPTER 5 | RUNNING EXPERIMENTS
The Startle Reflex system allows investigators to set up programs for performing basic startle
reflex experiments as well as Prepulse Inhibition of Startle (PPI) and Fear-Potentiated Startle
(FPS) experiments.
Sample Experiments
This chapter presents walkthrough examples of programming both PPI and FPS experiments in
mice, and will serve to familiarize the user with the process of setting up and running such
experiments. While the parameters used in these examples are based on guidelines devised by
Dr. William A. Falls in the Psychology Department at the University of Vermont, Burlington, VT
(see published references at the end of this chapter) they may not be suitable for the user’s
particular application. The user should conduct their own research to determine the appropriate
parameters needed for their field of study.
Glossary of Im portant Terms
Acclimation Duration: The period (in minutes) during which the program will be running but no
stimuli are presented and no data is collected.
Block: A phase of the experiment. An experiment contains up to 3 blocks. A block can contain
up to 100 trials. An experiment that contains logical sequential components (eg. Training or
Testing) can be separated into corresponding blocks.
Background Noise: A constant source of sound that is not the main stimulus. Background noise
is optional during acclimation.
Experiment Table: Is used to configure the exact trial sequence and parameters for each trial.
See Figure 5-5.
Inter-Trial Interval: The time (in seconds) from the end of the previous trial to the beginning of
the next trial.
Null Period: Time period prior to any stimulus presentation during which data is collected. Data
collected during the Null period determines whether the startle response is valid; in many trials,
the subject moves prior to the startle stimulus presentation. Movement in the Null period will
invalidate the response latency and amplitude in the ensuing Startle period.
Prepulse/Startle Delay: The time period (in milliseconds) between the onset of the prepulse and
startle stimulus. There is a maximum of 1000 milliseconds (see Figure 5-4).
Prepulse Stimulus Duration: In prepulse inhibition of startle (PPI), the period (in milliseconds)
during which a noise burst is presented prior to the startle stimulus.
Prepulse Stimulus Rise/Fall Time: Length of time (in milliseconds) for prepulse tone to rise to
and fall from the maximum dB level; used to suppress pops in the speaker when there is a
sudden change in signal.
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Pre-Stimulus 2 Record: Duration of recording time prior to Stimulus 2. Similar to the Null Period,
data collected during this period determines whether the startle response is valid.
Startle Period: The period (in milliseconds) during which the loud noise burst is presented.
Startle Stimulus Duration: The duration (in milliseconds) of the Startle Period.
Stimulus 1 Duration: In fear-potentiated startle (FPS), the duration of the Conditioned Stimulus
tone.
Stimulus 2 Duration: In fear-potentiated startle (FPS), the duration of the aversive stimulus.
Data Acquisition Period: The amount of time that data will be recorded during each trial.
Tone: Sine wave that is produced at the specified frequency and volume.
White Noise: Sound containing many frequencies at the same intensity.
Prepulse Inhibiti on (PPI)
Prepulse inhibition (PPI) of the startle response is a phenomenon in which a short pulse of
sound presented just prior to a startle stimulus (such as a burst of white noise at a high decibel
level) dampens the subsequent startle response.
This phenomenon has been observed in mice, rats and humans. When studied in rodents, PPI
serves as a model for a number of disorders, including deficits in attention/sensory gating or
sensorineural hearing loss.
Figure 5-1 - Prepulse Inhibition (PPI)
W.A. Falls 2003
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PPI Timing
Figure 5-2 illustrates the framework for one trial of the sample PPI experiment that will be
described in detail in the next section.
Figure 5-2 - Startle Reflex Timeline for PPI
Definitions of each time period depicted in Figure 5-2 are as follows:
(A) Acclimation = The period (in minutes) during which the program is running, but no
stimuli are presented and no data are collected.
(B) Data Acquisition Period = User-set value that defines the time period in which data
are recorded. It consists of the Null Period, the Prepulse/Startle Delay and the
Startle Period.
Note: Start with Data Acquisition Period (Figure 5-3) at 1000 ms to ensure that data are
collected after the startle stimulus is presented.
(C) Null Period = User-set value used to establish a baseline of the animal’s current
behavior. The Null Period is 200 ms in the example timeline above.
(D) Prepulse Stimulus Duration = User-set value that is the length of the prepulse tone. A
20 ms prepulse tone is used in the example above.
(E) Prepulse/Startle Delay = User-set value that is the time between the onset of the
prepulse stimulus and the onset of the startle stimulus, see Figure 5-1. The
Prepulse/Startle Delay must be greater than, or equal to the Prepulse Stimulus
Duration. In the example above the Prepulse/Startle Delay is set to 100 ms.
(F) Startle Stimulus Duration = User-set value that determines the duration of the noise
burst startle stimulus (Figure 5-5). It is set to 40 ms in the example above.
(G) Startle Period = The Startle Period begins at the start of the Startle Stimulus. The
Startle Period duration is equal to the Data Acquisition Period minus the Null
Period minus the Prepulse/Startle Delay. The Startle Period would be 700 ms in
the example.
Prepulse Inhibiti on (PPI) Sam ple Experiment
1.
Open the startle software and select File > New from the menu. This will open an
Experiment Configuration screen (See Figure 5-3). This screen is used to establish the
initial settings for the experiment, including Experiment Title, selection of Chamber(s) to be
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used, Acclimation Duration and Data Acquisition Period, number of Trials per Block, length
of Inter-Trial Interval (ITI), and a variable or fixed ITI.
Changes to this configuration can be made later by selecting Config/Experiment Config
from the menu.
Figure 5-3 - Experiment Configuration (PPI)
a. Enter the name of the experiment in the Experiment Title field;
b. In the Chambers field:
i.
Select the chambers to be used in the experiment;
ii.
Select the Background Noise Lockon box;
iii.
Enter 62 in the Background Noise Level (dB) box;
c. In the General Configuration field:
i.
Enter 5 in the Acclimation Duration (min) box;
ii.
Enter 1000 in the Data Acquisition Period (ms) box;
d. In the Configuration – Block (#) field, the buttons marked 1, 2, and 3 correspond to
different blocks, or phases, of the experiment. PPI has 3 phases and therefore
information will be configured for Block 1, Block 2 and Block 3, with respect to number
of trials per block, the ITI type and ITI Duration.
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Block 1 represents an initial phase, consisting of 10 trials in which the startle-eliciting
noise burst stimulus is presented alone (i.e. with NO prepulse), to determine a baseline
response to a startle stimulus.
Block 2 is the actual PPI testing phase that has 28 trials. Four trials consist of the noise
burst startle stimulus alone, 16 trials consist of a pre-pulse stimulus (at one of 4
different intensities) followed by the noise burst startle stimulus, four trials of prepulse
alone (one at each intensity) and four null trials (no stimulus presented).
Block 3 is the same as Block 1 in order to ensure that no habituation has taken place.
i.
To Configure Block 1: click on the 1 button, making sure the field title reads
“Configuration – Block 1”; enter 10 in the Trials box, choose ‘Variable’ from the
Inter-Trial Interval Type drop down menu and enter 10 and 20 in the Inter-Trial
Interval (sec) boxes;
ii.
To Configure Block 2: click on the 2 button, making sure the field title reads
“Configuration – Block 2”; enter 28 in the Trials box, choose “Variable” from the
Inter-Trial Interval Type drop down menu and enter 10 and 20 in the Inter-Trial
Interval (sec) boxes;
iii.
To Configure Block 3: click on the 3 button, making sure the field title reads
“Configuration – Block 3”; enter 10 in the Trials box, choose ‘Variable’ from the
Inter-Trial Interval Type drop down menu and enter 10 to 20 in the Inter-Trial
Interval (sec) box.
e. Entering a name in the Table Name field will not be done in this window, it will be done
at a later step in which an Experiment Table is set up, and will be the default table for
this example experiment.
f.
In the Comments field, enter any information that will be helpful in identifying the
parameters of this experiment.
g. Click OK and a “Save As” screen may appear. Click the Cancel button, as this
information will be saved at a later step. The information will not be lost.
2.
Select Config > Chamber Config from the menu. This will open a small “Chamber Config”
window and “Chamber (#)” will be highlighted; click OK. This will open a Chamber
Configuration screen, with “Chamber (#)” in the header, as shown in Figure 5-4. This
screen is used to set up individual chamber parameters for the different blocks, or phases,
of the PPI experiment.
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Figure 5-4 - Chamber Configuration Screen for PPI
a. Choose “Prepulse Startle” from the Experiment Type drop-down menu;
b. In the ID/Comment field, enter any pertinent information, such as animal info, etc.
c. In the Acclimation field:
i)
Do not change the default settings, shown above.
d. In the Startle Stimulus field:
i)
Choose “White Noise” from the Stimulus Type drop down menus;
ii) Enter 40 in the three Duration (ms) boxes;
iii) Enter 120 in the three Level (dB) boxes;
iv) Enter 1 in the three Rise/Fall Time (ms) boxes.
e. In the Prepulse Stimulus field:
i)
Enable only Block 2 by checking the Block 2 box in the Enabled section, and
unchecking Blocks 1 and 3;
ii) Select “White Noise” from the Stimulus Type drop down menu;
iii) Enter 20 in the Duration (ms) box;
iv) Leave the default value in the Level (dB) box at this point; the order of the varying
intensities will be set up later in these instructions;
v) Enter 1 in the Rise/Fall Time (ms) box;
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In the General field:
i)
Enter 200 in the Null Period (ms) box for all three blocks;
ii) Enter 100 in the Prepulse/Startle Delay (ms) box for block 2.
g. Click the Configure All Chambers with this Config button and click OK in the
confirmation window, then click OK in the lower left corner of the Chamber
Configuration window.
3.
Select Config > Experiment Table from the menu bar to open the Experiment Table screen
shown in Figure 5-5. For this example, all values for Blocks 1 and 3 were set in the chamber
configuration screen, only Block 2 needs to be configured. Use the selection bars on the left
to select Block 2.
Figure 5-5 - Experiment Table Screen (PPI)
a. Ensure the Null Period (ms) is set to 200 and the Prepulse/Startle Delay (ms) is set to
100;
b. Ensure the Prepulse Enabled and Startle Enabled check boxes are checked;
c. Enter the individual trial parameters for Block 2 outlined in Table 6-1. The actual PPI
experiment is set up in Block 2 and consists of 28 pseudo-randomized trials pairing
different decibels of prepulse stimulus or no prepulse stimulus with the noise burst
startle stimulus.
d. In the Set All… field, select Chambers and click the Modify button;
e. Click on the Save Table button, choose a file name and save the table;
f.
Click the Exit button on the ‘Stimulus Control Table’ window.
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Next select Config > Experiment Config from the menu. This will open the Experiment
Configuration window (Figure 5-3).
a. Click on the Select Table button to find and select the experiment table that was just set
up, click Open, then OK on the Experiment Configuration window.
b. Finally, select File > Save Config from the menu bar and choose a name for this
program. It will be saved with an .exp extension.
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Block 2
Block 1
Table 5-1 – Manually Entered Trial Parameters for PPI
Stimulus Type
Duration (ms)
PP
S
PP
S
PP
S
PP
S
Trial Type
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
5
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
6
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
7
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
8
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
9
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
10
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
1
√
√
WN
WN
20
40
67
120
1
1
Pre-pulse + Startle
2
Trial
Pre-pulse
Enabled
Startle
Enabled
1
Un-√
2
3
4
Level (dB)
Rise/Fall Time (ms)
Un-√
Un-√
-
-
-
-
-
-
-
-
Null
3
√
Un-√
WN
-
20
-
70
-
1
-
Pre-pulse Only
4
√
√
WN
WN
20
40
73
120
1
1
Pre-pulse + Startle
5
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
6
√
√
WN
WN
20
40
70
120
1
1
Pre-pulse + Startle
7
√
√
WN
WN
20
40
76
120
1
1
Pre-pulse + Startle
8
Un-√
Un-√
-
-
-
-
-
-
-
-
Null
9
√
√
WN
WN
20
40
73
120
1
1
Pre-pulse + Startle
WN
20
40
67
120
1
1
Pre-pulse + Startle
Pre-pulse Only
10
√
√
WN
11
√
Un-√
WN
-
20
-
76
-
1
-
12
√
√
WN
WN
20
40
70
120
1
1
Pre-pulse + Startle
13
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
14
√
√
WN
WN
20
40
76
120
1
1
Pre-pulse + Startle
15
√
√
WN
WN
20
40
67
120
1
1
Pre-pulse + Startle
16
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
17
√
√
WN
WN
20
40
76
120
1
1
Pre-pulse + Startle
18
Un-√
Un-√
-
-
-
-
-
-
-
-
Null
WN
20
40
73
120
1
1
Pre-pulse + Startle
19
√
√
WN
20
√
Un-√
WN
-
20
-
67
-
1
-
Pre-pulse Only
21
√
√
WN
WN
20
40
70
120
1
1
Pre-pulse + Startle
22
√
Un-√
WN
-
20
-
73
-
1
-
Pre-pulse Only
23
√
√
WN
WN
20
40
70
120
1
1
Pre-pulse + Startle
24
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
25
√
√
WN
WN
20
40
76
120
1
1
Pre-pulse + Startle
26
Un-√
Un-√
-
-
-
-
-
-
-
-
Null
27
√
√
WN
WN
20
40
67
120
1
1
Pre-pulse + Startle
28
√
√
WN
WN
20
40
73
120
1
1
Pre-pulse + Startle
WN = White Noise; PP = Pre-pulse; S = Startle
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Stimulus Type
Duration (ms)
PP
S
PP
S
PP
S
PP
S
Trial Type
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
4
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
5
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
6
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
7
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
8
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
9
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
10
Un-√
√
-
WN
-
40
-
120
-
1
Startle Stimulus Only
Trial
Pre-pulse
Enabled
Startle
Enabled
1
Un-√
2
Un-√
3
Level (dB)
Rise/Fall Time (ms)
WN = White Noise; PP = Pre-pulse; S = Startle
Special Options Utility
In the lower left corner of the Chamber Configuration window (Config > Chamber Config) is a
“Special Options” button. When this button is clicked, it brings up a Special Options screen
(Figure 5-6) with “Startle Reflex Special Options” in the header. This option allows the user to
vary levels for the Prepulse and Startle stimuli in Prepulse Inhibition experiments.
Figure 5-6 - Startle Reflex Special Options
If only the Varying Prepulse Level radio button is selected, the program will increment the
Prepulse Level on each trial as set in the Minimum Level, Maximum Level, and Step Value
boxes for the relevant Blocks. For example, using the values in Figure 5-6, for a Prepulse
Inhibition program, for Block 1, the first trial would have a Prepulse Level of 70 dB, the next trial
would have a Prepulse with a value 5 dB higher (75 dB) and so on, until trial 5, which would
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reach the maximum value set, 90 dB. If there are more than 5 trials in Block 1, the Prepulse
Level will return to 70 dB and continue to cycle through the settings as just described.
If only the Varying Startle Level radio button is selected, the Startle Level will vary according to
the settings depicted in Figure 5-6, in the same fashion as described for “Varying Prepulse
Level”.
If both the Varying Prepulse Level and Varying Startle Level are selected for the settings
depicted in Figure 5-6, the trials begin with the minimum Level Prepulse and Startle Levels and
increment through all the Prepulse Levels as set, then it will increment to the next Startle Level
and again, increment through all the Prepulse Levels as set. For example: the first trial in Block
1 will have a Prepulse Level of 70 dB and a Startle Level of 100 dB, the second trial will have a
Prepulse Level of 75 dB and a Startle Level of 100 dB…the fifth trial with have a Prepulse Level of
90 dB (Maximum Level set) and the startle Level will still be 100 dB. The sixth trial the Prepulse
Level will be at the Minimum Level setting, 70 dB while the Startle Level will be at the next
increment level of 110 dB. Trials 7 – 10 will have the Prepulse Level increment as just described,
with the Startle Level remaining at 110 dB. This pattern will continue until trial fifteen, when the
Prepulse Level and the Startle Levels will reach the Maximum Levels set: 90 dB and 120 dB,
respectively. If there are more than 15 trials, the pattern will repeat, starting with the Minimum
Level values set.
If a combination of varying and constant blocks are desired, set the varying blocks as described
above and set constant blocks by entering “0” in the Step Value boxes for the blocks that should
be kept constant. If a Stimulus in a block should be disabled, enter “0” in the Maximum Level
and Minimum Level boxes for the appropriate block.
When finished setting values in the Special Options window, click the OK button. This saves the
settings. If “Varying Prepulse Level” has been selected, the Level (dB) box in the “Prepulse
Startle” field of the associated Chamber Configuration Window will be “grayed-out”. If
“Varying Startle Level” was selected, then the same will be true for the Level (dB) boxes in the
“Startle Stimulus” field of the associated Chamber Configuration Window.
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Fear -Potentiated Star tle (FPS)
Fear-Potentiated startle (FPS) is a phenomenon in which the startle response to an acoustic
stimulus is increased (potentiated) in the presence of a Conditioned Stimulus (CS). The CS is a
normally neutral stimulus (usually a tone) that has been introduced together with an aversive
stimulus (a shock) during training. This Pavlovian conditioning is a learned response.
FPS studies begin with a training protocol, consisting of a simple pairing of a tone with an
aversive stimulus. Once training is complete, testing is performed. Testing consists of the
presentation of the CS tone with the white noise burst startle stimulus at pseudo-random
intervals.
The fear-potentiated acoustic startle reflex is observed in both rodents and humans, thus
making it a useful tool for investigating learning and memory, including long-term memory.
Both FPS training and FPS testing sample experiments are described in detail below (from W.A.
Falls 2002).
Figure 5-7 - Fear-Potentiated Startle (FPS)
W.A. Falls 2002
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FPS Timing
Figure 5-8 - Startle Reflex Timeline for Potentiated Experiments
Definitions of the time periods in Figure 5-8 :
(A) Acclimation = The period (in minutes) during which the program is running, but no
stimuli are presented and no data are collected.
(B) Data Acquisition Period = A user-set value that defines the time period in which data
is recorded. It consists of the Null Period, the Pre-Stimulus 2 Record Period, and the
Stimulus 2 Period.
Note: Start with a Data Acquisition Period (Figure 5-9) of 1000 ms to ensure that data is
collected after the startle stimulus is presented.
(C) Null Period = A user-set value used to establish a baseline of the animal’s current
behavior. The Null Period is 200 ms in the example timeline above.
(D) Stimulus 1 Duration = A user-set value that defines the duration of the Conditioned
Stimulus tone, set to 30000 ms in the sample timeline.
(E) Pre-Stimulus 2 Record = A user-set value that defines the duration of the Duration of
recording time prior to Stimulus 2, set to 200 ms in the sample timeline.
Note: Data recorded during Pre-stimulus 2 Record will be defined as “Stimulus 1 Time
Period” when saving data later.
(F) Stimulus 2 Duration = A user-set value that defines the duration of the Aversive
Stimulus, set to 250 ms in the sample timeline.
(G) Stimulus 2 Period = A system-calculated value that is equal to the Data Acquisition
Period (1000 ms) minus the Null Period (200 ms) minus the Pre-Stimulus 2
Record (200 ms) = 600 ms in the sample timeline above.
(H) Inter-Trial Interval = Latency between successive trials. May be variable or fixed.
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Fear -Potentiated Star tle (FPS) Traini ng Sample Experiment
Open the Startle Reflex software and select File > New. This will open the Experiment
Configuration window, shown in Figure 5-9. This window is used to define the initial
settings for the experiment, such as experiment title, chamber(s) to be used, length of
acclimation, Data Acquisition Period, number of trials per block and length of inter-trial
interval (ITI), as well as choosing a variable or fixed ITI. Changes to this configuration may
be made later by selecting Config > Experiment Config.
1.
NOTE: Do not use the Hardware > Training option to set up this training program, see Chapter 4
for use of this utility.
Figure 5-9 - Experiment Configuration Screen for FPS
a.
Enter a name for the experiment in the Experiment Title field;
b. In the Chambers field:
i)
Select the chambers to be used in the experiment;
ii) De-select (uncheck) the Background Noise Lockon box;
c. In the General Configuration field:
i)
Enter 5 in the Acclimation Duration (min) box (this is the period during which the
program will be running but no stimuli are presented);
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ii) Enter 1000 in the Data Acquisition Period (ms) box;
d. In the Configuration – Block (#) field,
i)
The buttons marked 1, 2, and 3 are to select different blocks, or phases, of the
experiment. FPS training has only 1 phase and therefore information will be
configured for Block 1 with respect to number of trials per block, the ITI type and
duration. Training will consist of a 5 min acclimation period (set up in previous step)
followed by 10 trials in which the Conditioned Stimulus (CS), a tone (70 dB; 12000
Hz) lasting 30 seconds, is paired with the unconditioned stimulus, a 0.4 mA shock
lasting 0.25 second. Pairings will be presented at a variable interval.
ii) To Configure Block 1:
(1) Click on the 1 button, make sure the field title reads “Configuration – Block 1”;
(2) Enter 10 in the Trials box;
(3) Select ‘Variable’ from the Inter-Trial Interval Type drop down menu;
(4) Enter 90 and 180 in the Inter-Trial Interval (sec) boxes.
e. If the Table Name field is not set to “default.ta$”, select the “default.ta$” table.
f.
Enter any information that will be helpful to identify the parameters of this experiment
in the Comments field;
g. Click OK and a ‘Save As’ box will pop-up, provide a meaningful experiment name and
click Save.
2.
Next, Select Config > Chamber Config to open the “Chamber Config” screen, “Chamber
(#)” will be highlighted; click OK. The Chamber Configuration screen, Figure 5-10, is used
to set up individual chamber parameters for the different blocks, or phases, of the FPS
experiment (FPS training has only 1 phase).
a. Select ‘Potentiated Startle’ from the Experiment Type drop down menu;
b. Enter any pertinent information, such as animal info, etc. in the ID/Comment field;
c. No steps are required in the Acclimation field.
d. In the Stimulus 2 field:
i)
Verify that ‘Auxiliary w/ Stimulus 1’ is selected from the Stimulus Type drop down
menu;
ii) Enter 250 in the Duration (ms) box;
iii) Enter the auxiliary port number on the PHM-255A that the ENV-414S unit is
connected to.
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Figure 5-10 - Chamber Configuration (FPS)
e. In the Stimulus 1 field:
i)
Enter 30000 in the Duration (ms) box;
ii) Enter 70 in the Level (dB) box;
iii) Enter 3 in the Rise/Fall Time (ms) box;
iv) Enter 12000 in the Frequency (Hz) box.
f.
In the General field:
i)
Enter 0 in the Background Noise (dB) box;
ii) Enter 200 in the Null Period (ms) box;
iii) Enter 200 in the Pre-stimulus 2 Record (ms) box.
g. Click Configure All Chambers with this Config, click on OK in pop-up window, and then
click OK button in lower left corner of the Chamber Configuration window.
h. Click OK to exit the Chamber Configuration screen.
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Fear -Potentiated Star tle (FPS) Testing Sample Experiment
The following is a sample FPS testing experiment. Such a testing experiment would normally
follow the preceding training component.
1. Open the startle software and select File > New.
Configuration window (see Figure 5-9).
This will open an Experiment
a. Enter a name for the experiment in the Experiment Title field;
b. In the Chambers field:
i)
Select the chambers to be used in the experiment;
ii) De-select (uncheck) the Background Noise Lockon box;
c. In the General Configuration field:
i)
Enter 5 in the Acclimation Duration (min) box (this is the period during which the
program will be running but no stimuli are presented);
ii) Enter 1000 in the Data Acquisition Period (ms) box;
iii) This FPS testing experiment example will have two phases and therefore
information will need to be configured for Block 1 and Block 2. Block 1 will be a 9
trial leader sequence phase to establish a stable startle baseline. The trials will
consist of 3 presentations of stimuli at 3 different volumes in a pseudo-random
order, without presentation of the conditioned stimulus (CS) tone. Block 2 has 3
different noise burst startle stimuli, each presented 6 times. Half of the block 2 trials
will present a CS tone.
iv) To Configure Block 1:
(1) In the “Configuration Block (#)” field, click on the 1 button, be sure the field title
reads “Configuration – Block 1”;
(2) Enter 9 in the Trials box;
(3) Select ‘Fixed’ from the Inter-Trial Interval Type drop-down menu;
(4) Enter 60 in the Inter-Trial Interval (sec) box.
v) To Configure Block 2:
(1) Click on the 2 button, be sure the field title reads “Configuration – Block 2”;
(2) Enter 18 in the Trials box;
(3) Select ‘Fixed’ from the Inter-Trial Interval Type drop-down menu;
(4) Enter 60 in the Inter-Trial Interval (sec) box.
d. Again, selecting a name in the “Table Name” field will not be done in the Experiment
Configuration window, it will be done at a later step in which an Experiment Table is set
up, and will be the default table for this example experiment.
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e. In the Comments field, enter any information that will be helpful to identify the
parameters of this experiment.
f.
Click OK and a “Save As” box will pop-up, provide a meaningful experiment title and
click Save.
2. Next, Select Config > Chamber Config. This will open a small “Chamber Config” window and
“Chamber (#)” will be highlighted; click OK. This will open a Chamber Configuration
window, with “Chamber (#)” in the header (see Figure 5-11).
Figure 5-11 - FPS Testing Chamber Configuration Window
a. Verify that ‘Potentiated Startle’ from the Experiment Type drop down menu;
b. Enter any pertinent information, such as animal info, etc. in the ID/Comment field
c. No steps are required in the Acclimation field.
d. In the Stimulus 2 field:
i)
Enable Block 1 and Block 2 by selecting both Block 1 and Block 2 boxes in the
Enabled section;
ii) For Block 1, choose ‘White Noise’ from the Stimulus Type drop down menu and for
Block 2, choose ‘White Noise w/ Stimulus 1’ from the drop down menu;
iii) Enter 30 in the Duration (ms) box for both Blocks 1 and 2;
iv) Enter 110 in the Level (dB) box for both Blocks 1 and 2 and 3 in the Rise/Fall Time
(ms) box for both Blocks 1 and 2.
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e. In the Stimulus 1 field:
i)
Enable only Block 2 by checking the Block 2 box in the Enabled section, and uncheck Block 1;
ii) Select ‘Pure Tone’ from the Stimulus Type pull down menu;
iii) Enter 30000 in the Duration (ms) box;
iv) Enter 70 in the Level (dB) box;
v) Enter 3 in the Rise/Fall Time (ms) box;
vi) Enter 12000 in the Frequency (Hz) box.
f.
In the General field:
i)
Enter 200 in the Null Period (ms) box for both blocks;
ii) For Block 1 enter 0 in the Pre-stimulus 2 Record (ms) box and enter 200 for Block 2.
g. Click the Configure All Chambers with this Config button, then click on OK in the
confirmation window;
h. Click OK in lower left corner of the Chamber Configuration window.
3. Next, select Config > Experiment Table. This will open an Experiment Table screen (Figure
5-12), with “Stimulus Control Table – default.ta$.” in the header. The Experiment Table
screen is where the exact trial sequence and parameters for each trial will be set up. In the
upper left corner of the window are selection bars to be used to advance through blocks and
trials.
Figure 5-12 - Experiment Table Window
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4. Ensure that the appropriate Block and Trial # appear above the selection bars for the Block
and trial being set up;
5. Enter 200 in the Null Period (ms) box;
6. Verify that the Pre-stimulus 2 Record (ms) box has 0 for both Block 1 and Block 2;
7. Block 1, the Leader Sequence phase, is used to establish a stable startle baseline. In this
block the 3 sets of the 3 intensities of noise burst startle stimuli (‘Stimulus 2’) are presented.
Check only the Stimulus 2 checkbox and enter the values from Table 5-2.
Table 5-2 - Trial Parameters for Block 1 (Leader Sequence Phase)
Stimulus 1
Trial #
Stimulus 2
Type
Duration (ms) Level (dB)
Rise/Fall
Time (ms)
Frequency (Hz)
Type
Duration (ms) Level (dB)
Rise/Fall
Time (ms)
1
NA
NA
NA
NA
NA
WN
30
105
3
2
NA
NA
NA
NA
NA
WN
30
110
3
3
NA
NA
NA
NA
NA
WN
30
100
3
4
NA
NA
NA
NA
NA
WN
30
110
3
5
NA
NA
NA
NA
NA
WN
30
100
3
6
NA
NA
NA
NA
NA
WN
30
105
3
7
NA
NA
NA
NA
NA
WN
30
100
3
8
NA
NA
NA
NA
NA
WN
30
105
3
9
NA
NA
NA
NA
NA
WN
30
110
3
NA = Not Applicable
WN = White Noise
8. For entering individual Trial Parameters for Block 2, enter the values from Table 5-3. Trials
that have a stimulus 1, highlighted rows in Table 5-3, need to have a Pre-stimulus 2 Record
of 200 ms. See Figure 5-13.
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Figure 5-13 - FPS Testing Block 2 Experiment Table Window
Table 5-3 - Trial Parameters for Block 2 (Testing Phase)
Stimulus 1
Trial #
Type
Stimulus 2
Duration
Level
Rise/Fall
Frequency
(ms)
(dB)
Time (ms)
(Hz)
Type
Duration
Level
(ms)
(dB)
Rise/Fall
Time (ms)
1
PT
30000
70
3
12000
WN
30
105
3
2
NA
NA
NA
NA
NA
WN
30
110
3
3
PT
30000
70
3
12000
WN
30
100
3
4
NA
NA
NA
NA
NA
WN
30
100
3
5
PT
30000
70
3
12000
WN
30
110
3
6
NA
NA
NA
NA
NA
WN
30
105
3
7
NA
NA
NA
NA
NA
WN
30
110
3
8
PT
30000
70
3
12000
WN
30
110
3
9
NA
NA
NA
NA
NA
WN
30
100
3
10
PT
30000
70
3
12000
WN
30
105
3
11
PT
30000
70
3
12000
WN
30
100
3
12
NA
NA
NA
NA
NA
WN
30
105
3
13
PT
30000
70
3
12000
WN
30
100
3
14
NA
NA
NA
NA
NA
WN
30
105
3
15
PT
30000
70
3
12000
WN
30
110
3
16
NA
NA
NA
NA
NA
WN
30
100
3
17
NA
NA
NA
NA
NA
WN
30
110
3
18
PT
30000
70
3
12000
WN
30
105
3
NA = Not Applicable
PT = Pure Tone
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9. In the “Set All…” field, select Chambers and click the Modify button.
10. Click on the Save Table button and choose a file name and save the table; click the Exit
button on the ‘Stimulus Control Table’ window.
11. Next select Config > Experiment Config. This will open the Experiment Configuration
window.
12. Click on Select Table, find and select the experiment table that was just set-up, click Open;
click the OK button of the Experiment Configuration window.
13. Finally, select File > Save Config from the menu bar and choose a name for this program (it
will be saved with an .exp extension).
Running PPI or FPS Experiments
To run experimental sessions with the PPI program or either the training or testing FPS
programs outlined in this chapter, select File > Load Config and select the appropriate .EXP file.
Once the experiment file is open, click Run > Begin Experiment.
Saving Data fr om PPI or FPS Experiments
At the conclusion of each experiment it is important to click File > Save Data to open the “Save
As” window and provide a unique data file name for each experiment’s data. If a unique data
file name is not supplied, the previous experiment’s data will be overwritten.
After saving the data file see Chapter 7, Data Management, for other data saving and exporting
options.
Refer ences
For PPI:
Jaworski DM, Boone J, Caterina J, Soloway P, Falls WA. (2005). Prepulse inhibition and
fear-potentiated startle are altered in tissue inhibitor of metalloproteinase-2 (TIMP-2)
knockout mice. Brain Research. Volume 1051, Issues 1-2, 27 July; 81-89.
For FPS:
Falls, W.A. (2002). Fear-Potentiated Startle in Mice, Current Protocols in Neuroscience
Supplement 19, Unit 8.11B; J. Crawley, Editor, John Wiley & Sons.
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CHAPTER 6 | MENU SELECTIONS
File Menu Options
New: Closes the current experiment and brings up the Experiment Configuration screen.
Load Config: Allows the user to load pre-determined Experiment Configuration parameters so
that new parameters do not have to be entered every time an experiment is run.
Save Config: Saves the current configuration to an .EXP Experiment Configuration file. It is
recommended that the Experiment Configuration files be named to reflect the protocols
(configuration) that they apply to. This will allow the user to recognize and use these files at
a later time.
Save As Default Config: Makes the current Experiment Configuration file the default
configuration to be loaded when the Startle Reflex software begins.
Print Config: Prints experiment and chamber configurations either to a text file or to the default
Windows printer.
Save Data: Stores data collected from the current experiment for future analysis with the
Startle Reflex software. When saving data, Startle Reflex generates a .DAT file and a .EXP
file with the same name as the .DAT file. This .EXP file is used by Startle Reflex when
opening .DAT files during analysis and must not be confused with the .EXP Experiment
Configuration file generated when Save Config is selected. The .EXP file generated when
saving session data cannot be used as an Experiment Configuration file for running an
experiment. It is recommend that .DAT and .EXP data files be stored in a separate folder
from the Experiment Configuration .EXP files to avoid confusion between file types.
Load Data: Retrieves and displays data files saved from a previous experiment. Specify the
experiment filename either with its .DAT extension or with no extension.
Save Data as…: Allows the user to save raw or analyzed data in a standard ASCII format for use
in third party spreadsheet or database software. For detailed information on exporting data
files see Chapter 7, Data Management. For information on displaying and adjusting data
display see the Data Menu Options section of this chapter.
Print Data: Displays the Print Data dialog box. Specify sets and ranges of data to print to the
default Windows printer. The Print Data dialog functions identically to the Save as ASCII
dialog described in the previous section.
Exit: Exits the program. If an experiment has been run, but not saved, the user is prompted to
save the current experiment and data file.
Hardwar e Menu Options
Configure Hardware: Allows the user to select which amplifier is to be used. Click the Configure
ADC Device button to check the status of the DIG-744 data acquisition card, see Figure 6-2.
Please note, Startle Reflex will use the first device listed on the ADC Device Properties screen.
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The ADC Device Properties screen enables the user to name, test, detect and remove the DIG744 data acquisition card(s) as desired.
Figure 6-1 - Hardware Configuration Screen
Figure 6-2 - ADC Device Properties Screen
Calibrate Audio: For the speakers to generate reproducible audio startle stimuli, audio
calibration must be performed. The audio calibration should be performed each time the
audio values used in protocols are changed. This requires the measurement of output
volume over a range of frequencies at specific volume levels. This information is then saved
to a data file that will ensure accurate audio output levels during subsequent experiments.
See Chapter 5, Calibration for audio and input calibration procedures.
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Stimulus Test: The Stimulus Test utility is used to test attachment of all audio and auxiliary
stimuli (Figure 6-3).
Figure 6-3 - Stimulus Test Screen
Menu options for testing stimulus outputs:
Table 6-1 - Stimulus Test Options
Option
Description
Chamber
Enter the number of the chamber to test.
should remain “1”.
Stimulus Type
Select a Stimulus Type (Pure Tone, White Noise, or any of the four Auxiliary
outputs).
Frequency (Hz)
Enter a test frequency of 100 to 32,767 Hz in 1 Hz increments.
Rise/Fall Time (ms)
Enter a Rise/Fall Time of 0 to 250 ms in 1 ms increments.
Duration (ms)
Enter a stimulus Duration of 1 to 250 ms in 1 ms increments.
Stimulus Level (dB)
Enter a Startle pulse level of 40 to 130 dB in 1 dB increments.
Background Level (dB)
Enter a Background noise level of 30 to 100 dB in 1 dB increments.
Pulse Stimulus
Create the specified Startle pulse.
Stimulus Lock
Click these buttons to lock on and turn off the Startle stimulus output in the
chamber.
Background Noise Lock
Click these buttons to lock on and turn off the background noise speaker in the
chamber.
Exit
Returns to the main program.
If using the ANL-925D, this number
Calibrate Input: Use this menu option to test or fine-tune the sensitivity of the Startle Reflex
platform hardware. See Chapter 5, Calibration for audio and input calibration procedures.
Training: Use this menu option to condition an animal to expect an aversive stimulus after a
Pure Tone stimulus. The duration for the Auxiliary stimulus (shock) can be longer than 250
ms during training. See Figure 6-4.
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Figure 6-4 – Training Screen
Table 6-2 - Training Options
Option
Description
Trials
The number of trials for this training session. The valid range is 1 to 100
trials.
Inter-Trial Interval (sec)
Enter an Inter-Trial Interval in seconds. The Inter-Trial Interval is the time
from the end of the previous trial to the beginning of the next trial. The
valid range is 1 to 999 seconds in 1-second intervals.
Background Noise (dB)
Enter the Background Noise level to be used during the trial period. The
valid range is from 30 to 100 dB.
Acclimation Duration (min)
Enter the duration of the acclimation period in minutes. The valid range is
from 0 to 99.0 in 0.1 min increments.
Enable Acclimation Auxiliary
Check this box to have Auxiliary stimulus during the acclimation period.
Acclimation Auxiliary Port
If the acclimation auxiliary is selected, enter the auxiliary port number.
Valid ports are 1 to 4.
Enable Acclimation
Background Noise
Check this box to have background noise during the acclimation period.
Background Noise Level (dB)
If Background Noise is selected, enter the dB level of the noise. The valid
range is from 30 to 100 dB.
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Option
Description
Stimulus Type
Select Pure Tone, White Noise or Auxiliary. The Stimulus 2 may also include
the Stimulus 1 signal, if they are not conflicting signals. Note: For Stimulus 2
the Auxiliary or Auxiliary w/Stimulus 1 option should be selected for aversive
stimulus presentation.
Duration (ms)
Enter a stimulus duration from 1 to 32767 ms in 1 ms increments.
Level (dB)
Enter an audio stimulus level from 40 to 130 dB in 1 dB increments.
Rise/Fall Time (ms)
Rise/Fall Time is used to suppress pops in the speaker when there is a
sudden change in the signal. Enter a Rise/Fall Time of 0 to 50ms in 1ms
increments.
Frequency
Enter a Pure Tone Frequency value from 100 to 32,767 Hz in 1 Hz
increments.
Auxiliary Port
Enter the port number to be turned on during an auxiliary stimulus. Valid
ports are 1 to 4.
Load
The Load button is used to retrieve a saved training setup from a file.
Specify the training filename with its .TRN extension or with no extension.
Save
The Save button is used to save any training setup to a binary file. Specify
the file with its .TRN extension or with no extension. Filenames with no
extension receive the .TRN extension by default.
OK
Start the training session.
Cancel
Closes the Training windows or if a training session has already been started,
it cancels the training session and closes the training window.
Config Menu Opti ons
The Config Menu selections determine experiment and data configurations. These are
described in greater detail in the Sample Experiments section of Chapter 5. The Config
and Experiment Table menu items are active once an experiment is loaded.
Experiment Config: Use this option to display and edit the global experiment configuration
options. These settings can be modified at any time prior to running an experiment.
Chamber Config: After a chamber is selected, the Chamber Configuration screen for that
chamber is displayed. The Chamber Config screen may also be accessed by clicking the
Config button located in the Chamber 1 display of the open experiment. This screen is used
to set up individual chamber parameters for the different blocks, or phases, of an
experiment.
Chamber Data: Select the chamber to edit and then click OK, or click the Data button in the
Chamber 1 display of the open experiment configuration to open the Chamber Data screen,
see Figure 6-5. Use the Chamber Data screen to add additional experiment information.
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Figure 6-5 - Chamber Data Screen
Experiment Table: This screen is where the exact trial sequence and parameters for each trial
can be set up. In the upper left corner of the screen are selection bars to be used to
advance through blocks and trials. Any changes made to this table may affect the
Experiment Configuration and Chamber Configuration data.
Data Menu Options
Options: The Data Options screen (Figure 6-6) is used to adjust how data will be saved to your
text file under the Computed Values option (see File Menu Selections earlier in this chapter).
See Table 6-3 for a list of parameters that can be adjusted in the data file.
Figure 6-6 - Data Options
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Table 6-3 - Data Options
Option
Description
Startle
Response Only
The Total and Average values computed are based on the first positive wave that
meets the minimum wave criteria. The area between lines A and B in Figure 6-7.
Startle and
Platform
Response
The Total and Average values computed are based on the first positive wave that
meets the minimum wave criteria and the following negative wave (“peak-to-peak” or
“peak-to-trough” detection).The area between lines A and C in Figure 6-7.
First Peak
Largest Peak
Reports the Peak Time and Peak Values for either the first data peak or the largest
data peak.
Peak Data
Points
Gives the Duration, Total and Average for either all of the data points or only the
peak data points.
All Data Points
Minimum
Latency (ms)
This value is used when searching for the startle response. If the start of a wave (ie.
the first positive value after a negative value) occurs before the minimum latency
time, it is not counted as the startle response.
Minimum Peak
Value
This value is used when searching for the startle response. If a peak occurs below the
minimum peak value, it is not counted as the startle response.
Minimum Peak
Time (ms)
This value is used when searching for the startle response. If a peak occurs before
the minimum peak time, it is not counted as the startle response.
OK
Save all changes and return to the main screen.
Cancel
Ignore all changes and return to the main screen.
Load Data Options: Load Data Options is used to retrieve previously saved data options from a
file. Specify the data options filename with its .DA$ extension or with no extension.
Save Data Options: Save Data Options is used to save the data options setup to a binary file.
Once you have made changes to Data Options screen, you must then Save Data Options to
make the changes effective. Specify the file with its .DA$ extension or with no extension.
Filenames with no extension receive the .DA$ extension by default.
Figure 6-7 - Startle and Platform Response Example
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Run Menu Opti ons
Begin Experiment: When the experiment and chamber configurations have been completed,
select this option to begin the experiment.
Abort Experiment: Choose this menu option if a running experiment must be aborted prior to
completion. Data collected from completed trials will not be lost and can be saved.
Post Analysis: Select this option to view and analyze data (Figure 6-8).
Figure 6-8 - Post Analysis
Table 6-4 - Post Analysis Options
Option
Description
Chamber
Use these scroll bars to specify which chamber, block and trial to view. The
data and graphs will be displayed accordingly.
Block
Trial
Overlay
Click this button to superimpose the current graph over the graph display.
This feature allows the user to directly compare two or more graphs. To
clear the graph, click Refresh.
Mark Peak
Click this button to display the peak time and value on the graph. Click this
button again or click Refresh to clear the display.
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Option
Description
Refresh
Click this button to clear and redraw the current graph. Selecting this
button also has the effect of clearing any superimposed graphs displayed
using the Overlay button.
Graph Options
Click this button to display the graph options dialog box. Refer to Graph
Options in the following section.
Print Screen
Click this button to print the screen to your default Windows printer.
Data Options
Click this button to display the Data Options dialog box.
Options in the previous section.
Load Data Windows
Click this button to load the raw Null, Prepulse, and Startle time period data
of a particular trial into display windows.
Exit
Select this button to return to the main program.
Refer to Data
Graph Menu Opti ons
Print Active: Prints the active window to the default Windows printer.
Refresh All: Refreshes all the chamber display windows. Refer to Refresh Active described
below.
Refresh Active: Use this option to redraw the graph and calculated data for the active window.
This is useful if chamber graph windows become cluttered or erased when another window
passes over a graph.
Options: Displays the Graph Options dialog (Figure 6-9).
Figure 6-9 - Graph Options
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Table 6-5 - Graph Options
Option
Description
Plot Style
Select either line graph, bar graph, or scatter plot.
Maximum Y Axis Value
Specify the largest Y value desired on the graphs. Graphs will be scaled
accordingly.
Enable Runtime Display
Only disable Runtime Display when running experiments with exceptionally
short Trial Intervals. Disabling the runtime display ensures the most
accurate interval timing possible. If the user specifies a Trial Interval that
may be too short to enable runtime graphing, the program will
automatically prompt the user and disable runtime graphing.
Enable Period Marking
Lines
Turn on or off the vertical marking lines that clearly mark the starting and
stopping points of the Null Period, Stimulus 1 Period and the Stimulus 2
Period.
OK
Save all changes and return to the main screen.
Cancel
Ignore all changes and return to the main screen.
Window Menu Options
Cascade: Arranges the windows in a cascade fashion.
Tile: Arranges the windows in a tile fashion.
Arrange Icons: Orders the windows that have been reduced to icons in the bottom of the
screen, from left to right.
About Menu Selecti on
This displays the Startle Reflex copyright and version screen.
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CHAPTER 7 | SAVING AND EXPORTING DATA
At the conclusion of each experiment it is important to click File > Save Data to open the “Save
As” window and provide a unique data file name for each experiment’s data. If a unique data
file name is not supplied, the previous experiment’s data will be overwritten.
After the data file has been saved, Startle Reflex offers different options for exporting the data
for use in spreadsheet and database applications. With the experiment’s data open in Startle
Reflex click File > Save Data as … to access the following options.
Computed Values
The Computed Values option, Figure 7-1, saves the data as an annotated or stripped text file.
The annotated format is recommend as it includes the experiment information while the
stripped format does not. The Computed Values option also includes the option to save the data
into a Microsoft Access® database file (*.mdb file) by selecting the Database File radio button.
See the Opening A Startle Database With Excel section for information on importing the *.mdb
file into Microsoft Excel™. The user may also select to only export a portion of the data file by
exporting the data for only selected chambers, blocks or trials. See Table 7-1 for more
information.
Figure 7-1 - Computed Values Export Option
Raw Data
The Raw Data export option, Figure 7-2, exports the actual raw, unprocessed data to a text file.
The Raw Data option allows the user to export in the annotated (recommended) or stripped
format, select which data sampling (time periods) period(s) to export, and select which
chambers, blocks and trials to export. See Table 7-1 and Importing a Text Data File Into Excel
for more information.
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Figure 7-2 – Raw Data Export Option
Statistical Data
Figure 7-3 illustrates the Statistical Data export option. Similar to the other export options, the
user can select an annotated or stripped data file format, select which chambers, blocks and
trials to export, and it provides the option to calculated statistical values such as averages,
standard deviation and standard error of the mean. See also Table 7-1 and Importing a Text
Data File Into Excel.
Figure 7-3 - Save Data As Option With Statistical Values
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Table 7-1 - Save Data As Export Options
Option
Description
Computed Values
Choose Computed Values to save a text file containing latency to startle,
peak time, peak value, duration, total and average for each sample period.
Raw Data
Select Raw Data to save actual unprocessed data values. See Chapter 9,
Startle Units Explained.
Statistical Data
Select Statistical Data to save averages, standard deviation, and standard
error of the mean for each sample period.
Null Period
Prepulse Time Period
Startle Time Period
If Raw Data has been selected, check the desired sample period(s) to save to
disk.
Averages
Standard Deviation
Standard Error of the Mean
If Statistical Data has been selected, check the desired statistic(s) to save to
disk.
Annotated File
Annotated files include file information and headings.
Stripped File
Select Use Tab Separators to insert a tab between each field instead of a
space. In order to open a Tab Separated file, right click on the file name and
select Open with Excel.
Database File
The Database File format saves data in a Microsoft Access® Database (*.mdb)
file, and is only available if the Computed Values option was selected.
Chambers
Specify the chamber or range of chambers to save as ASCII. Specify the
bottom of the range first, followed by the top of the range.
For example: Chambers 3 to 6.
Blocks
Specify the block or range of blocks to save as ASCII. If the user has
selected to run only 1 block of trials then block 1 should be selected,
otherwise, specify the bottom of the range first, followed by the top of the
range.
For example: Blocks 1 to 2.
Trials
Specify the trial or range of trials to save as ASCII. Specify the bottom of
the range first, followed by the top of the range.
For example: Trials 45 to 73.
Samples
If Raw Data has been selected, specify the sample or range of samples to
save as ASCII. Specify the bottom of the range first, followed by the top of
the range. For example: Samples 100-250.
Note: The maximum range value must be equal to or less than the number
of samples.
Save As
Brings up the Save As file dialog. Enter the desired filename.
Cancel
Returns to the main menu without saving the data to disk.
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Opening A Startl e Database With Excel
If the Computed Values export option was used to save the data as a Microsoft Access®
database file (*.mdb), it can be opened in Microsoft Excel™ by using the instructions
below to create a query to import the data.
Start Microsoft Excel™. Click the Data tab and in the Get External Data section click the
drop down arrow for From Other Sources and select From Microsoft Query as shown in
Figure 7-4.
Figure 7-4 - New Database Query
Select MS Access Database from the list of databases (Figure 7-5), and click the OK
button.
Figure 7-5 - Choose Data Source
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Navigate to the Startle directory and select the desired database as shown in Figure 7-6
and click OK.
Figure 7-6 - Select Database
Click Trial Data and then the > button to add all of the database columns to the Columns
in your query: list (Figure 7-7). Click on an item in the Columns in your query: list and
click < to remove it from the query. If unsure, include them all, as fields may be deleted
later. Select an item and use the up and down arrows on the right to change the order in
which the items appear. Click Next.
Figure 7-7 - Query Wizard
If the message shown in Figure 7-8 appears, click the OK button.
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Figure 7-8 - Microsoft Query Message
The next display, as shown in Figure 7-9, allows the data to be filtered to only present
certain data. Leave blank to display all data. Click Next.
Figure 7-9 - Filter Data Query Wizard
The data may then be sorted based on multiple criteria, see Figure 7-10 . Click Next.
Figure 7-10 - Sort Order
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On the Finish screen select Return Data to Microsoft Office Excel and click Finish or
Save Query to save the query for reuse and then Finish. Refer to Figure 7-11.
Figure 7-11 - Finish Screen
By saving the query, it will be able to be rerun in Excel at a later date, with a new
database. The only time these steps will need to be gone through again is if the data
fields returned to Excel need to be modified.
Excel will now prompt the user (Figure 7-12) for a location on the spreadsheet to place
the returned data. Click on the OK button to open the data in Excel.
Figure 7-12 - Returning External Data to Microsoft Excel
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Figure 7-13 shows what the returned data looks like in Excel.
Figure 7-13 - Queried Data in Excel from MS Access Database
To run a saved query, go to Data > Get External Data > From Microsoft Query as shown
in Figure 7-4.
Select the saved query from the Queries tab as shown in Figure 7-14 and click the Open
button.
Figure 7-14 - Selecting Saved Query to Run
Select the data table(s) to work with, the filter(s) to apply and the sort order to use
as shown in Figure 7-7.
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Importing a Text Data File Into Excel
A spreadsheet program may be used to open data saved as an ASCII text file. This section
illustrates how to open ASCII data using Microsoft ExcelTM. The Use Tab Separators option must
be used when saving the data for these steps to work.
1. In Excel, select File > Open. Locate where the file has been saved. Select All Files (*.*) in
the file type drop down menu.
2. Select the desired data file to open and click Open.
3. This will open step 1 of the Text Import Wizard, Figure 7-15. Be sure Delimited is selected
and click Next. In step 2 of the import wizard, Figure 7-16, check the box for Tab Delimiters
and click Finish.
Figure 7-15 - Text Import Wizard Step 1
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Figure 7-16 - Text Import Wizard Step 2
4. The data will appear in Excel in different formats depending on how the data was exported.
The format will vary depending on whether a stripped or annotated file was saved and
whether computed values, raw data or statistical data was exported. See the File Formats
section for examples.
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File Formats
Sample PPI experiment raw data saved in the stripped format:
1
(chamber)
1
(block)
1
(trial)
[NEWLINE]
Condition: Startle Stimulus
[NEWLINE]
23
(Null Time Period - First value)
345
(Null Time Period - Second value)
234
(Null Time Period - Third value)
[NEWLINE]
23
(Prepulse Time Period - First value)
345
(Prepulse Time Period - Second value)
234
(Prepulse Time Period - Third value)
[NEWLINE]
23
(Startle Time Period - First value)
345
(Startle Time Period - Second value)
234
(Startle Time Period - Third value)
Sample computed values saved in the stripped format:
1
1
[NEWLINE]
95
122
120
52
3816
73.4
55
78
88
43
2327
54.1
37
55
457
32
9479
296.2
21
22
[NEWLINE]
[NEWLINE]
2
1
[NEWLINE]
0
0
(chamber)
(block)
(Latency to Startle - Trial 1 - Null Time Period)
(Peak Time - Trial 1 - Null Time Period)
(Peak Value - Trial 1 - Null Time Period)
(Startle Duration - Trial 1 - Null Time Period)
(Total - Trial 1 - Null Time Period)
(Average - Trial 1 - Null Time Period)
(Latency to Startle - Trial 1 - Prepulse Time Period)
(Peak Time - Trial 1 - Prepulse Time Period)
(Peak Value - Trial 1 - Prepulse Time Period)
(Startle Duration - Trial 1 - Prepulse Time Period)
(Total - Trial 1 - Prepulse Time Period)
(Average - Trial 1 - Prepulse Time Period)
(Latency to Startle - Trial 1 - Startle Time Period)
(Peak Time - Trial 1 - Startle Time Period)
(Peak Value - Trial 1 - Startle Time Period)
(Startle Duration - Trial 1 - Startle Time Period)
(Total - Trial 1 - Startle Time Period)
(Average - Trial 1 - Startle Time Period)
(Latency to Startle - Trial 1 - Null Time Period)
(Peak Time - Trial 1 - Null Time Period)
(chamber)
(block)
(Latency to Startle - Trial 2 - Null Time Period)
(Peak Time - Trial 2 - Null Time Period)
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CHAPTER 8 | ALTERNATE CALIBRATE INPUT UTILITY
The Calibrate Input utility is used to adjust the sensitivity of the response platform using the
spinner and solenoid accessories offered for older systems.
Figure 8-1 - Calibrate Input Screen for Spinner Calibrators
Figure 8-2 - Calibrate Input Screen for Solenoid Calibrators
Menu options for input calibration:
Table 8-1 - Calibrate Input Options
Option
Description
Chamber
Enter the chamber number of the platform to be calibrated.
Calibrator Type
Select the type of calibrator being used.
Output Level (dB)
If speaker calibrator is selected, enter the output level for the calibration woofer. The
default value is 75 dB, but a smaller value may be used if calibrating the system for
mice.
Frequency (Hz)
If the speaker calibrator is selected, enter the pulse frequency for the calibration
woofer. This value must fall within the response range of both the speaker and
platform. 10 Hz works well.
Begin
This button turns on the calibrator while sampling and graphing data. Observe and
modify the Startle Platform Amplifier gain until it is at the appropriate level for the
experiment.
End
Stop the calibrator and data collection graphing.
Done
Stops data collection and graphing. Returns the user to the main program.
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The response platform generates an analog signal that is digitized and brought into the
software. A preamplifier boosts the signal before it reaches the analog to digital converter in
the computer. The gain of the preamplifier must be set to produce a signal in the desired
amplitude range.
The Calibrate Input Utility is used to simulate the pressure generated on the platform by an
animal's response by placing the calibrator on the response platform, securing it firmly to the
platform with the thumbscrews, and driving it with the appropriate signal. The resulting signal
is displayed on the screen. Adjusting the gain of the amplifier affects the signal's amplitude. It is
important to have the platform responses calibrated as closely as possible in order to have
consistent amplitude measures between subjects. The trick is to not only have equal amplitudes
between platforms for a given force, but to maximize the response, within the dynamic range of
the system, without clipping (off the scale, so to speak) the signal. This provides accuracy and
resolution.
To run the calibration procedure,open Startle Reflex and click File > New from the main menu.
Specify the chambers to be calibrated by selecting their check boxes in the upper left hand
corner of the Experiment Configuration window, see Figure 5-3, and click the OK button. An
experiment that is already set up with the desired number of chambers may also be loaded.
When the chamber display windows have been loaded, select Hardware > Calibrate Input from
the main menu.
Begin by entering the chamber number to calibrate in the Chamber box. Next, select the type of
calibrator that is going to be used. Place the calibrator on the response platform. Connect the
calibrator's cable to the AUX 1 port on the PHM-255A in the selected Chamber.
Click the Begin button. The calibrator applies a 10 Hz modulated force to the platform and the
software plots the response data. Observe the data. The response data window displays the
average Positive Peak value and the average Negative Peak value of ten responses. Adjust the
amplifier gain so that the average amplitude is at the desired level. When satisfied with the
platform’s gain adjustment, use the positive peak value as a standard for calibrating all the other
platforms. The gain settings between amplifiers should be close. If the amplifier settings vary
considerably, please contact Med Associates, Inc.
NOTE: The value that is chosen to calibrate to is dependent upon the subject types and the
resolving power that should be obtained. We suggest that some pre-study information be
gathered, such as the average amplitude of a CD - 1 male mouse startle with the platform gain
set at 5, or the average amplitude of a Sprague-Dawley rat with the platform gain set to 0.1.
The important thing is to set all platforms to the same value and maintain this value throughout
the duration of the protocol.
Repeat this procedure for each additional chamber. When all chambers have been calibrated,
click the Done button on the Calibrate Input dialog box.
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CHAPTER 9 | STARTLE UNITS EXPLAINED
Researchers studying the acoustic startle reflex often wonder what units their data should be
expressed in. Most commercial systems designed to measure the acoustic startle reflex use a
sensor to convert the animal’s movement into a digital signal that is analyzed by the computer.
Various methods exist for calibrating such sensors, and various types of sensors can be used, but
usually startle responses are quantified in terms of “Arbitrary Units,” “Startle Units,” “Startle
Response”, “A/D Units,” or some other similar designation (e.g. Bortolato et al, 2007; Jaworski
et al, 2005; Meloni et al, 2006; Risbrough & Geyer, 2005; Vinkers et al, 2007; Winslow, Noble, &
Davis, 2007). The within-subjects design of startle experiments indicates that such modes of
quantitation are usually acceptable, independent of the specific vendor of the startle apparatus.
The most important consideration for the researcher is that a standard calibration procedure be
adopted that ensures similar sensitivities among all the startle sensors in his/her system.
Q: What are the units for data gathered using SOF-825 Startle Reflex software?
We are often asked what units the actual data from the startle sensor are expressed in. Startle
Reflex software utilizes a highly sensitive analog-to-digital converter that converts the analog
voltage signal from the startle sensor to a digital unit having a value between –2048 and +2048.
The converter can handle voltages between –10 Volts and +10 Volts. In most cases, people
simply leave their startle responses expressed on a scale of 0 to 2048 “Arbitrary Units” or
“Startle Response Units” (e.g. Bortolato et al, 2007; Jaworski et al, 2005; Meloni et al, 2006).
However, these values can be easily converted to a dimensioned quantity, such as Voltage.
Recalling the principles of operation described above, you can simply divide your startle
response by 2048, and multiply this number by 10.
This process would yield the actual voltage measured by the analog-to-digital converter. This
value can serve to instill confidence that similar sensitivities are achieved across all of your
startle chambers. However, it is still difficult to compare actual data from different publications,
unless you know specific calibration procedures that were used to generate the data of interest.
We advocate publishing as much detail as possible regarding calibration methods. This allows
researchers in different labs to make the most informative conclusions about the reported data,
regardless of the units that the data are expressed in.
References:
Bortolato M, Frau R, Orru M, Piras AP, Fa M, Tuveri A, Puligheddu M, Gessa GL, Castelli MP,
Mereu G, Marrosu F. Activation of GABA(B) receptors reverses spontaneous gating deficits in
juvenile DBA/2J mice. Psychopharmacology (Berl). 2007 Jun 29; DOI 10.1007/s00213-007-08455.
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Jaworski DM, Boone J, Caterina J, Soloway P, Falls WA. Prepulse inhibition and fear-potentiated
startle are altered in tissue inhibitor of metalloproteinase-2 (TIMP-2) knockout mice. Brain Res.
1051(1-2):81-89, 2005.
Meloni EG, Jackson A, Gerety LP, Cohen BM, Carlezon WA. Role of the bed nucleus of the stria
terminalis (BST) in the expression of conditioned fear. Ann. N. Y. Acad. Sci. 1071:538-541, 2006.
Risbrough VB, Geyer MA. Anxiogenic treatments do not increase fear-potentiated startle in
mice. Biol. Psychiatry. 57(1):33-43, 2005.
Vinkers CH, Risbrough VB, Geyer MA, Caldwell S, Low MJ, Hauger RL. Role of dopamine D1 and
D2 receptors in CRF-induced disruption of sensorimotor gating. Pharmacol. Biochem. Behav.
86(3): 550-558, 2007.
Winslow JT, Noble PL, Davis M. Modulation of fear-potentiated startle and vocalizations in
juvenile rhesus monkeys by morphine, diazepam, and buspirone. Biol. Psychiatry. 61(3):389-395,
2007.
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CHAPTER 10 | CONTACT INFORMATION
Please contact Med Associates, Inc. for information regarding any of our products.
Visit our website at www.med-associates.com for contact information.
For technical questions, call 802-527-2343 or email support@med-associates.com.
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