Michael Kinder provides insight on the underlying technologies used in startle systems and explains what researchers should know about sensor capabilities and limitations and how that knowledge can help create effective protocols, address calibration and properly analyze data. The startle reflex in rodents is typically studied using startle chambers with integrated transducers to measure elicited responses to aversive stimuli. The importance of startle systems in pre-clinical research labs is evident based on the pervasive use of this technology in both academia and industry for applications such as behavioral phenotyping, pre-pulse inhibition studies and safety pharmacology and toxicology screening. As is true with any scientific instrument, understanding the underlying technology and inherent limitations is important when designing experimental protocols, setting up and calibrating the equipment and analyzing data. Michael Kinder, System Designer at Kinder Scientific, presents advancements in modern startle systems that scientists should know. Topics include: - What are the technologies available that scientists need to understand? - What are the physical characteristics of the sensory assemblies? - Does subject weight matter in startle response studies? - Should scientists be comparing raw startle amplitude between animals of different sizes? - Does the unit of measure matter? - What does an analog only trial provide (or not provide)?

1. Advancements in
Modern Startle
Response Systems
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3. Advancements in Startle Reflex:
An in-depth discussion on piezo/Plexiglas
sensors versus load cell sensors
Michael Kinder
President, CEO
Kinder Scientific Company
VP Business Development
OtoScience Labs
Copyright 2015 Michael Kinder, Kinder Scientific and InsideScientific. All rights reserved.

4. Thank you to our event sponsor
 Open Field
 Cage Rack
 Pulse Maze
 Rotometer
 Light / Dark
 Startle Reflex
 Forced Swim
 Active / Passive
Avoidance
 Tail Suspension
 Cue and Contextual Fear
Conditioning
 Place Preference
 Learning Hole board
Kinder Scientific -
Behavioral System Core

5. Today’s discussion
1. A history of piezo/Plexiglas sensing assemblies
and why it’s time to move on:
• What does it actually measure
• Is it a true accelerometer?
• Strengths & Weaknesses of the design
2. The applicability of Load Cell sensors:
• Strengths &Weaknesses

6. The high speed videos in this presentation
have been provided by:
Alexander Galazyuk, Ph.D.
Associate Professor
Department of Anatomy & Neurobiology
Northeast Ohio Medical University
We are very grateful for Alex’s generosity and stunning quality
of work that he and Ryan Longenecker demonstrated in the
creation of these videos.

7. What measure are
we after ?
A Whole Body Reflex from a loud noise!

9. For over 20 years the piezo/Plexiglas
sensing assembly was the dominant method
for measuring startle responses
Much of the science on startle reflex stands
on the shoulders of this technique.

10. How the piezo/Plexiglas sensor works
Spring alone
(no energy)
Subject’s mass Subject’s mass + Response
• Because the Plexiglas
plate is a spring, the signal
coming off the piezo
necessarily contains the
spring characteristics
• This adds significant and
sometimes confounding
complexity to analyzing
the data

11. • The design requires some
method of centering the
subjects downward force
over the piezo
• It is important to
understand that the piezo is
the transducer, not the
sensor. The entire
assembly is the sensor (or
sensor assembly)
How the piezo/Plexiglas sensor works
Piezo glued to plate
and sealed
Platform legsPlexiglas plate
Subject reflex response

12. • Animal mass preloads the
spring of the Plexiglas
plate
• Think if this as stored
energy what will be
released back into the
data
How the piezo/Plexiglas sensor works
Plexiglas/Piezo Sensor
Animal Mass

13. • When the animal startles,
the Plexiglas spring is now
loaded with the animal
mass + the startle
response
• This is why the second
positive peak can be
larger than the first
How the piezo/Plexiglas sensor works
Subject reflex response bends the plate
Plexiglas/Piezo Sensor

14. Early Waveforms…25 years ago
Graph of – 115d20msHabit @ Trial 8
• This early approach
provides only the
absolute values and
makes it very difficult
to know what is
actually happening
from a study of the
waveform.

15. Early Improvements to Full Wave Signal!
Graph of – 115d20msHabit @ Trial 8
• This is the exact
animal data from the
previous slide.
• It is clear that the
“spring” is oscillating
• And the sensing
assembly is not
faithfully following
the animal response

17. • What does the output of the sensing assembly actually represent?
– The combination of the response and the spring!
• Is it truly an accelerometer?
– No! this is a misnomer. It is more of a kinetic energy measurement.
• Does the animal weight matter?
– Yes! Dampens Spring
• Can it be properly calibrated?
– Possibly, but with great difficulty.
Has the piezo/Plexiglas sensor outlived
its usefulness?

18. Calibrating the piezo/Plexiglas sensor
– or is it only equilibrating?
• The piezo transducer produces a DC voltage analogous to the
amount of bend it experiences.
• However, because it bleeds off the signal so quickly, it only outputs
a value when the plate is in motion. Likely here is where some of
the confusion over being an accelerometer. But the voltage it
creates is not analogous to acceleration but instead change in
distance (bend).

19. • Calibration - verb (used with object), calibrated, calibrating. 1.
to determine, check, or rectify the graduation of (any instrument
giving quantitative measurements).
• Because we are trying to measure the force exuded by the animal,
“calibrating” in volts is not actually calibrating. The best we can
hope for in this approach is “equilibration”, i.e., making the sensors
from multiple stations produce the same output value for a known
input force.
Equilibrating

20. Equilibrating the piezo/Plexiglas sensor with an
eccentric vibrating motor

21. Challenges & weaknesses with an eccentric
vibrating motor
1. The frequency difference between the Plexiglas plate and the revolutions
of the motor cause inconsistent forces to occur. They beat against each other.
2. Makes it difficult to calibrate to a standard unit of force such as Newton.
3. Because the animal weight has a significant effect on the spring
characteristics of the sensor, one would need a calibrator for each animal
weight equal to the animal weight. Therefore, it is not practical to pursue
true calibration, but rather, accept equilibration as the goal.
4. It is difficult to prove linearity

22. 1. We are stuck with using arbitrary units such as volts or
just a number or…
2. We can link the performance of the plate to a Newton with a single pulse
solenoid that is calibrated on a NIST traceable apparatus. Still, this does not
accurately tie the unit of measure to Newton because of the effect of the
animal weight.
3. This is why you should not compare raw amplitudes of different weight
animals, or even the same animal at different ages when using a
piezo/Plexiglas sensing assembly. Not apples and apples, but rather apples
and oranges.
Piezo/Plexiglas Sensors…

23. Moving to
the next level

24. Load Cell Sensor Assembly
Strengths:
• Faithfully follows the animal response
• Easy static calibration with fixed weights (easily made NIST
traceable)
• Easy to prove linearity
• Ability to compare responses of different weight subjects
Weaknesses:
• More expensive to manufacture

25. Load Cell
Sensor
Faithfully
follows
response

26. Post Session Trial Retrace with Load Cell Sensor
• Note the
response from
the system is very
similar to the
video. The
negative peak is a
result of the mass
of the restrainer
0.0
4.0N

27. It’s about
the data!

28. The Purpose of Nostim Trials
Is this the right question?
What is the purpose of Nostim (no stimulus) trials?
What was the subject doing during these times?

29. • What was the
subject doing just
prior to startle
elicitation?
• That is what
should determine
the validity of the
trial.
Evaluating pretrial activity
0.0
8.0N

30. 0.0
8.0N
Dual Analysis Windows
• Each trial can be
automatically
evaluated for
pre-startle
activity?
Window A Window B

31. 0.0
8.0N
Dual Analysis Windows
Window A
1. Determine True
Pre-startle activity
in the first window
2. Accurately set the
second window for
the first positive
response
Window B

32. Final Thoughts
• Dr. Alexander Galazyuk’s publication in the Journal of
Neuroscience Methods “An improved approach to separating
startle data from noise”
• Pay close attention to how your system is calibrated or equilibrated and
know the difference between the two. Make sure to continually convince
yourself that your system is recording the same response for a known input.
It is one of the most overlooked area of startle research.
• Ask yourself if comparing startle amplitudes of different size animals
matters to you and decide accordingly

33. Thank You!
For additional information on startle response systems,
sensor technologies, and best practices for calibration
and protocol design, please visit:
http://www.kinderscientific.com
Michael Kinder
michael@kinderscientific.com
(858) 679-1515

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information regarding innovative
technologies, protocols, research
tools and laboratory services.