Employing Acoustic, Tactile and PPI Startle Response Procedures in Rodent Behavioral Research
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Transcript of Employing Acoustic, Tactile and PPI Startle Response Procedures in Rodent Behavioral Research
![Page 1: Employing Acoustic, Tactile and PPI Startle Response Procedures in Rodent Behavioral Research](https://reader031.fdocuments.net/reader031/viewer/2022021922/586f752a1a28ab10258b5f33/html5/thumbnails/1.jpg)
Employing Acoustic, Tactile and PPI Startle Response Procedures in Rodent Behavioral Research
Dr. Charles Vorhees and Jason Adair discuss the value and methodology of Acoustic, Tactile and Pre-Pulse Inhibition paradigms for startle reflex testing in rodents…
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InsideScientific is an online educational environment designed for life science researchers. Our goal is to aid in
the sharing and distribution of scientific information regarding innovative technologies, protocols, research tools
and laboratory services.
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Employing Acoustic, Tactile and PPI Startle Response Procedures in Rodent Behavioral Research
Copyright 2016 San Diego Instruments and InsideScientific. All Rights Reserved.
Jason AdairSenior Engineer,San Diego Instruments
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1. no user restrictions from the testing software – flexibility to implement whatever startle paradigm they want
2. minimal subject stress from restraint or enclosure devices
3. ability to test multiple subjects simultaneously
4. accurate results regardless of the startle procedure/paradigm
Key components of an effective startle response system…
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Startle paradigms are created
via trials and sessions which
allow users to build a
chronological script of events.
SR-LAB™ - The smart, flexible choice for startle reflex testing
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The tubular animal enclosures are designed to allow the animals to turn around freely without constraint—thus avoiding restraint stress that can alter results. At the same time, the tubular design keeps animals over the sensor for reliable results.
5 different size animal enclosures
SR-LAB™ - The smart, flexible choice for startle reflex testing
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Test up to 16 stations
simultaneously
Animal enclosures can be
exchanged quickly to increase
throughput
SR-LAB™ - The smart, flexible choice for startle reflex testing
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SR-LAB’s accelerometer is a key
factor in the accuracy the
system provides. The sensor
reacts very quickly to sudden
force changes—assuring you
get an accurate reading for the
smallest mouse to the largest
rat.
SR-LAB™ - The smart, flexible choice for startle reflex testing
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• Complete system with sound attenuating cabinet
• Audio adjust dial
• Response adjust dial for sensitivity
• Adjustable doors to ensure good fit
• USB control unit
SR-LAB™ - The smart, flexible choice for startle reflex testing
To learn more about the SR-LAB System click here
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1. Acoustic Startle Response (ASR)
• To elicit a startle response from sudden acoustic stimuli
2. Tactile Startle Response (TSR)
• To elicit a startle response via air puffs onto the skin
3. Pre-Pulse Inhibition (PPI)
• Weak acoustic stimuli delivered prior to startle pulse inhibits startle reflex
Key terms for today’s lecture…
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Example of ASR trial…
• Broadband noise used to create background noise level
• Pulse has quick rise-time
• Unexpected
Acoustic Startle Response (ASR)
time (mS)
Am
plit
ud
e (
dB
)
Pulse
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Example of TSR trial…
• Short puffs of air are delivered to animal
• Adjustable up to 60 psi
• Can be used as pulse alone or in PPI
Tactile Startle Response (TSR)
time (mS)
Inte
nsity
Air Puff
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Example of pre-pulse inhibition trial…
• Broadband noise used to create uniform background level
• Typically prepulses are a few dB above background
• Pulse typically in the 110-120 dB range
Pre-Pulse Inhibition (PPI)
time (mS)
Am
plit
ud
e (
dB
)
Prepulse
Pulse
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• Access down to mS level for construction of your definitions
• Trials and sessions saved as discrete files
• Trial and session names carry through to data for future reference
Trial Definitions
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• Minimize variability between users by having pre-defined sessions that are ready to run
• Add/Insert/Delete trials
• Organize trials into blocks for protocol design and data sorting
• Define session once and use forever
Session Definitions
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• ITI number generator
• Enter ITIs manually
• Use as few or as many as you wish
• ITIs are saved and used in every session that is run. Every animal is exposed to the same structure.
Inter-Trial Intervals
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Diagnostics
• Important tool for consistent and accurate data
• Verify stability of SR-LAB cabinets
• Identify dB levels of interest (pp levels, startle pulse levels)
• Calibration of cabinet sensitivity
• Standardization across boxes
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• Data collected is shown real-time
• Every data point is stored in database
• Vstart, Vmax, Tmax, Avg
• Exportable to your stat package
csv
tsv
rows
columns
Data
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Data
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Data
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• Shock amplitude can be varied within trial or from one to the next
• 0-5 mA range
Foot Shock
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Tactile Kit
• Used with TSR
• The Tactile Kit provides an air puff stimulus, which is generally used as a kinder, gentler alternative to shock.
• 0-60 P.S.I.
Air Puff
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Pure Tone Kit
The Pure Tone Kit supplies up to three specific frequencies of sound and is generally used as a stimulus. This kit is also used for gap detection startle reflex testing.You control the frequency, amplitude and duration via the SR-LAB software.
PPI LED Light Kit
An add-on light to the animal enclosure is used as a Pre-Pulse stimulus to provide cross-modal startle reflex testing. On and off times are controlled from the SR-LAB software.
High Intensity Light Kit
The kit supplies an immense amount of bright light within the test station, which brings on anxiety in the animal. The LED lights give off no heat, which can affect the animal. This kit is used for anxiety potentiated startle testing.
DOWNLOADSR-LAB™ Data Sheet
SR-LAB™ Accessories
DOWNLOAD San Diego Instruments Product Catalogue
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Applying Startle Response Procedures
Copyright 2016 C.V Vorhees and InsideScientific. All Rights Reserved.
Charles V. Vorhees, PhD Professor, Div. of NeurologyCincinnati Children’s Research FoundationUniversity of Cincinnati
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This is startle
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and this…
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How startle is measured
and this…
Acoustic Startle Response (ASR) in a rat
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Diaphragm & forelimb contraction
• Body mass restricts dynamic range• Inertia effects retard sensitivity• Body mass must be used as covariate• Momentum effects cause negative inflection
How startle is measured
• No effect of body mass on dynamic range• High temporal resolution eliminates body mass effects• Covariate analysis seldom needed• High sensitivity minimizes momentum effects
Neck, diaphragm, forelimb
Method 1 - Load Cell Method 2 - Accelerometer
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From Gomez-Nieto et al. (2014). Front. Neurosci., 8: 216. Illustration of ASR trisynaptic pathway.
CRN = cochlear root neuron; PnC = giant neurons of the caudal pontine reticular nucleus (also called the n. reticularis pontis caudalis)
The Acoustic Startle Response (ASR) Circuit
Gomez-Nieto et al. (2014). Front. Neurosci., 8: 216.
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30
How the ASR circuit may relate to measured output
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Startle
Conceptual Actual
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Research Objectives
• Pyrethroids are widely used insecticides.
• They are divided into Type I and Type II. Both affect Voltage-sensitive sodium channels (VSSC).
Type I’s produce repetitive firing; Type II’s depolarization block.
• Type I’s increase and Type II’s decrease ASR.
• There are few data in young animals even though children are exposed more than adults
• Goal: determine the age-dependent sensitivity
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Acute EffectsDesign & Methods
• Type I = permethrin (PRM);Type II = deltamethrin (DLM)
• Gavage; once
• Test: 2, 4, 6, 8 h later
• Sprague-Dawley CD IGS rats (Charles River, strain #001)
• ASR: 5 min acclimation
• PRM 100 trials; DLM 50 trials per session
• ITI = 20 s
• stimulus = 115 dB SPL mixed frequency
• recording window = 100 ms
• Groups matched by ASR pretest
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• Type I pyrethroid
• Repetitive neuronal firing
• Increases ASR
• “T” syndrome = tremor (at high doses)
Permethrin (PRM)
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C
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F i g . 2 . A d u l t A S R a f t e r p e r m e t h r i n . I n c e a s e s i n A S R w e r e
s i g n i f i c a n t a t 2 , 4 , 6 , a n d 8 h . N = 1 2 / g r o u p . * P < 0 . 0 5 ;
* * P < 0 . 0 1 , * * * P < 0 . 0 0 1 .
* *
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PRM: adult rats
• Increases in ASR significant at 4, 6 and 8 hours (h)
• N = 12/group
• *P<0.05, **P<0.01, ***P<0.001
• No tremor
• ASR is sensitive to PRM
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ax
(m
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C o n t r o l
P e r m e t h r i n
*
A
2 4 6 8
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ax
(m
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**
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**
‡
B
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I
F i g . 1 . E f f e c t s o f P R M o n A S R i n P 1 5 , P 1 7 , a n d P 2 1 m a l e r a t s .
L i t t e r s = 4 - 5 / d o s e / a g e ; n = 8 - 2 1 p u p s / a g e / d o s e .
PRM at P15, 17 & 21
• Dose-range finding experiment
• Litters = 4-5/dose/age
• N = 8-21 pups/age/dose
• Visible tremor
• ASR sensitive to PRM at higher doses than in adults
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Final test of PRM at PND15
• N = 16 litters per dose; 1 male and 1 female per litter (i.e. 32 pups per dose)
• Visible tremor
• ASR more sensitive than adults but in the presence of tremor
• As tremor went up, ASR went down
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C
60
mg
/ kg
90
mg
/ kg
12
0 m
g/ k
g
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
P 6 0 P e r m e t h r i n A S R 2 h
G r o u p
Vm
ax
(m
V)
C
60
mg
/ kg
90
mg
/ kg
12
0 m
g/ k
g
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
P 6 0 P e r m e t h r i n A S R 4 h
G r o u p
Vm
ax
(m
V)
C
60
mg
/ kg
90
mg
/ kg
12
0 m
g/ k
g
0
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2 5 0 0
P 6 0 P e r m e t h r i n A S R 6 h
G r o u p
Vm
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(m
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C
60
mg
/ kg
90
mg
/ kg
12
0 m
g/ k
g
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1 5 0 0
2 0 0 0
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P 6 0 P e r m e t h r i n A S R 8 h
G r o u p
Vm
ax
(m
V)
A B
C D
F i g . 2 . A d u l t A S R a f t e r p e r m e t h r i n . I n c e a s e s i n A S R w e r e
s i g n i f i c a n t a t 2 , 4 , 6 , a n d 8 h . N = 1 2 / g r o u p . * P < 0 . 0 5 ;
* * P < 0 . 0 1 , * * * P < 0 . 0 0 1 .
* *
** *
*
*****
**
**
*
PRM increases then decreases ASR in P15 rats as effect gradually shifts from facilitation to toxicity at doses that only increase ASR in adults; hence young rats are more sensitive
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• Type II pyrethroid
• Cause depolarization block
• Decrease ASR
• “CS” syndrome (choreoathetosis & salivation)(at high doses)
Deltamethrin (DLM)
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DLM in adult rats on ASR
• N = 16/dose group
• Even at 25 mg/kg, few overt symptoms
• With 50 trials/day little habituation
• ASR sensitive to DLM
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DLM at PND15
• 16 litters per dose; with 1 male and 1 female per dose per litter (ie. 32 pups per dose)
• Severity of ASR decrease greater in young than adult rats
• Dose-dependent tremor
• ASR effect at doses lower than in adults
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At doses lower than in adults, DLM induces dose-dependent decreased ASR lasting greater than 8 hours post-exposure
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• First data of age-dependent and time-dependent effects of Type I and Type II pyrethroids in rats as young as P15.
• Young rats are several-fold more sensitive to than adults
• ASR allowed us to determine the time-course of effects
What we learned about pyrethroids
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• Test-retest stability of ASR is unique
whereas
• Locomotor tests shows marked habituation
• L&M tests show trial-dependent learning
• Schedule-controlled operant behavior requires training and cannot be done at PND15
What we learned about pyrethroids
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TSR: Rat
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• Tactile startle response (TSR)
• ASR vs. TSR
• Adult male SD rats
• 8 per group
• TSR response 2-3 times greater than ASR
ASR vs. TSROther Startle Methods
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ASR-TSR after DLM
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• Adult male SD rats
• Alternate 10 Trial Blocks Of Each Stimulus
• N = 12/group
• TSR showed clearer effect than ASR
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0 2 4 6 8
2 0 0
4 0 0
6 0 0
8 0 0
P N D 1 5 D L M A S R
H o u r
Vm
ax
(m
V)
C o n t r o l
2 m g / k g
*† †
C
0 2 4 6 8
2 0 0
4 0 0
6 0 0
8 0 0
P N D 1 5 D L M T S R
H o u r
Vm
ax
(m
V)
C o n t r o l
2 m g / k g
* ‡ ***
D
F i g u r e . C o m p a r i s o n o f A S R t o T S R i n t h e s a m e a n i m a l s i n t h e
s a m e t e s t s e s s i o n s i n t e r d i g i t a t e d i n a l t e r n a t i n g 1 0 t r i a l b l o c k s o f
a c o u s t i c a n d t a c t i l e ( a i r p u f f ) s t i m u l i . N = 1 2 / g r o u p
P15 DLM: ASR VS. TSR
Comparison of ASR to TSR in the same animals in the same test sessions interdigitated in alternating 10 trial blocks of acoustic and tactile (air puff) stimuli. N = 12/group
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1. 50 trials
2. 3 ITIs: 8, 20, or variable 8,12,16,20,24 (16 s avg.)
ITI (s) Mean SEM ANOVA, F(2,12) = 0.22 (NS)
8 103.15 26.15
20 123.36 26.15
var 101.61 26.15
Hence, ITI length made no significant difference.
What is the effect of Intertrial interval (ITI) length?
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• Vmax = maximum response amplitude per trial (mV)
• Vavg = average response amplitude per trial (mV)
• Across many experiments correlation coefficient is between Vmax and Vavg, r = 0.96-98
• Both provide nearly identical information
Which dependent measure should be used?
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1. 100 trials
2. Prepulse 70 ms before startle stimulus (onset to onset)
3. Mice: include trials with only highest PP to ensure it does not elicit a startle response
4. Mice: watch for non-responders
5. Some strains have progressive hearing loss
C57BL/6 micePPI: Mice
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Aim: linear inhibition curve
If use %inhibition, show basic data to prove that baseline is unaffected
07
37
78
2
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
P r e p u l s e i n t e n s i t y ( d B )V
ma
x (m
V )
S a l
C i t
***
* †
A
PPI: rats SD rats
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Conclusions
ASR
• Versatile
• Validated across species
• Detects facilitation or inhibition
• Habituation
• Reflex modification (PPI)
• Known pathway
TSR
• Stronger response
• Minimal habituation
• Reflex modification (?)
• Similar pathway
• Promising
Click here to view a publication list for Dr. Vorhees, including the research shared in this presentation
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Thank You!If you have questions for the presenters please contact them by email.
For additional information on the solutions presented in this webinar please visit:
www.sandiegoinstruments.com
Chip Vorhees, [email protected]
Jason [email protected]