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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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

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…

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

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

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

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

• 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

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…

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

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

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

• 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

• 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

• 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

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

• 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

Data

Data

• Shock amplitude can be varied within trial or from one to the next

• 0-5 mA range

Foot Shock

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

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

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

This is startle

and this…

How startle is measured

and this…

Acoustic Startle Response (ASR) in a rat

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

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.

30

How the ASR circuit may relate to measured output

Startle

Conceptual Actual

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

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

• Type I pyrethroid

• Repetitive neuronal firing

• Increases ASR

• “T” syndrome = tremor (at high doses)

Permethrin (PRM)

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

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 6 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 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: 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

2 4 6 8

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0 1 2 0 m g / k g P 1 5

T i m e ( h )

Vm

ax

(m

V)

C o n t r o l

P e r m e t h r i n

*

A

2 4 6 8

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0 1 5 0 m g / k g P 1 5

T i m e ( h )

Vm

ax

(m

V)

**

**

‡

**

‡

B

2 4 6 8

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0 1 8 0 m g / k g P 1 5

T i m e ( h )

Vm

ax

(m

V)

***

*

C

2 4 6 8

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0 1 2 0 m g / k g P 1 7

T i m e ( h )

Vm

ax

(m

V)

**

‡

D

2 4 6 8

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0 1 5 0 m g / k g P 1 7

T i m e ( h )

Vm

ax

(m

V)

**

*** *

E

2 4 6 8

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0 1 8 0 m g / k g P 1 7

T i m e ( h )

Vm

ax

(m

V)

**

*

F

2 4 6 8

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

1 6 0 0 1 2 0 m g / k g P 2 1

T i m e ( h )

Vm

ax

(m

V)

*

G

2 4 6 8

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

1 6 0 0

T i m e ( h )

Vm

ax

(m

V)

* ***

*

**

**

**

*

1 5 0 m g / k g P 2 1

‡

H

2 4 6 8

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

1 6 0 0 1 8 0 m g / k g P 2 1

T i m e ( h )

Vm

ax

(m

V)

****

*** *

***

*

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

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

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

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 6 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 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

• Type II pyrethroid

• Cause depolarization block

• Decrease ASR

• “CS” syndrome (choreoathetosis & salivation)(at high doses)

Deltamethrin (DLM)

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

41

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

At doses lower than in adults, DLM induces dose-dependent decreased ASR lasting greater than 8 hours post-exposure

• 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

• 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

TSR: Rat

• 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

ASR-TSR after DLM

47

• Adult male SD rats

• Alternate 10 Trial Blocks Of Each Stimulus

• N = 12/group

• TSR showed clearer effect than ASR

48

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

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?

• 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?

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

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

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

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, PhDCharles.Vorhees@cchmc.org

Jason Adairjason@sandiegoinstruments.com