See JAX Pipeline on the IMPC website for order of tasks for all mice tested

Project protocol: Acoustic startle and pre-pulse inhibition

• Introduction
• Study design
• Workflow
• Equipment, software and supplies
• Reagents and solutions
• Procedure
• Measurements
• Experimental and technical parameters

Introduction

The acoustic startle response is characterized by an exaggerated flinching response to an unexpected strong auditory stimulus (prepulse). This response can be attenuated when it is preceded by a weaker stimulus (prepulse) and is the principle underlying prepulse inhibition (PPI). PPI has been described in numerous species, including mice and humans and provides an operational measure of sensorimotor gating reflecting the ability of an animal to successfully integrate and inhibit sensory information.

Study design

Paradigm: Baseline study

Panel: Collaborative Cross

Number of strains: 18

Sex: Both

Age: 9-11 wks

Sample size: Seven mice per sex from at least two litters

Number of cohorts: 1

Workflow

Step	Description	Equipment	Data collected
1	Mice are acclimated to test room for at least 30 min	-	-
2	Acoustic startle and prepulse inhibition measured	Sound attenuating chamber, load cell platform, sound generator	ASR evoked by background noise, ASR at 74, 78, 82, 120 dB, PPI at 74, 78, 82 dB, PPI evoked by global prepulse stimulus, maximum slope for PPI

Equipment, software and supplies

• Acoustic startle and prepulse inhibition apparatus (San Diego Instruments SR-LAB ABS System) [See Experimental and technical parameters]
  • Outer sound attenuating chamber that serves to prevent external noise or vibrations from interfering with experiment (15"w x 14"d x 18"h)
  • Within the sound attenuating chamber, an animal holder rests upon a load cell platform that records the startle response
  • A sound generator and software regulate sound stimulation from the speakers (San Diego Instruments SR-LAB ABS SYSTEM - DESKTOP P/N 2325-0004)
  • Four such chambers are covered with a sound proof blanket to further prevent noise from interfering with the test
  • Software: SR LAB

  
  

Reagents and solutions

• 70% ethanol

 

Procedure

1. Mice are allowed to acclimate to the testing room for a minimum of 30 min. Take care not to stimulate the mouse before starting the experiment. Do not change the cage on the day of the experiment.
2. Ensure that all apparatuses are functioning correctly by calibrating the vibration and sound level at the beginning of each day.
3. Place each mouse onto a load cell platform and secure the door closed. Cover four chambers with sound proof blanket.
4. The session is initiated with a 5 min acclimation period (background noise 70 dB).
5. The session is continued by presentations of 8 different trial types, each of which should be presented 6 times in pseudorandom order, with an inter-trial interval (ITI) varying randomly between 10 and 20 s. The trials are:
   • Three different acoustic trials of 20 ms duration of white noise stimuli which are presented alone (PP1, PP2, PP3 (74, 78, 82 dB)).
   • Three different prepulse trials where a prepulse (74, 78, 82 dB) precedes the pulse (120 dB) by 100 ms (PP1 + pulse, PP2 + pulse, PP3 + pulse) to derive the prepulse inhibition response. The intensities of the prepulse should be kept at levels above the background noise (BN = 70 dB) that do not elicit a significant startle response on their own, being approximately 2-20 dB above BN.
   • Startle pulse trials where 120 dB/40 ms of white noise is presented alone.
   • No stimulus (NOSTIM) trials in which only background noise (70 dB) is presented to measure baseline movement of the animal in the chamber (the BN set will vary depending on the noise of the environment).
6. Startle response is recorded every ms for 65 ms after the onset of startle, i.e., 40-60 ms during the startle plus 25-40 ms after the startle ends; 65-100 ms from the end of the previous ITI for NOSTIM.
7. Wipe chambers clean with 70% ethanol and allow time to dry before additional testing.

 

Data collection: The maximal peak-to-peak amplitude is used to determine the acoustic startle response. Basal startle responses (S) and prepulse startles (PP-S) are calculated respectively as the average responses to the startle presented alone and the average responses to the combined prepulse-startle.

Data QC: The calibration of the sound and the movement sensors are important for obtaining valid test results and therefore must be routinely calibrated (at least monthly).

Formulas and derivations: The amount of prepulse inhibition is calculated as a percentage score for each acoustic prepulse type: %PPI = 100 x (S - PP-S)/S. The global level of PPI is also calculated as the mean %PPI for the different prepulse responses: 100 x [S-(PP1-S + PP2-S + PP3-S)/3]/S.

 

Measurements

LIMS field name	MPD variable name	Description	Units	Series	Primary measurement	Series order	MPD computed	Calculation	Method	Data type
Response amplitude - BN	ASR_BN	average acoustic startle response (ASR) evoked by background noise	amplitude						acoustic startle test	Float
Response amplitude - S	ASR_74	average acoustic startle response (ASR), adjusted for background noise, 74 dB sound pressure level		Yes	ASR_74	1			acoustic startle test	Float
-	ASR_78	average acoustic startle response (ASR), adjusted for background noise, 78 dB sound pressure level		Yes	ASR_74	2			acoustic startle test	Float
-	ASR_82	average acoustic startle response (ASR), adjusted for background noise, 82 dB sound pressure level		Yes	ASR_74	3			acoustic startle test	Float
-	ASR_120	average acoustic startle response (ASR), adjusted for background noise, 120 dB sound pressure level		Yes	ASR_74	4			acoustic startle test	Float
% PPI PP1	PPI_74	average percent prepulse inhibition (PPI) evoked by 120 db startle, adjusted for background noise, 74 dB prepulse	%	Yes	PPI_74	1		See Formulas and derivations (above)	acoustic startle test	Float
% PPI PP2	PPI_78	average percent prepulse inhibition (PPI) evoked by 120 db startle, adjusted for background noise, 78 dB prepulse	%	Yes	PPI_74	2		See Formulas and derivations (above)	acoustic startle test	Float
% PPI PP3	PPI_82	average percent prepulse inhibition (PPI) evoked by 120 db startle, adjusted for background noise, 82 dB prepulse	%	Yes	PPI_74	3		See Formulas and derivations (above)	acoustic startle test	Float
% PPI Global	PPI_global	average percent prepulse inhibition (PPI) evoked by global prepulse stimulus (74, 78, 82 dB), adjusted for background noise	%					See Formulas and derivations (above)	acoustic startle test	Float
-	PPI_slope	maximum slope for percent prepulse inhibition (PPI) averages (74, 78, 82 dB), adjusted for background noise							acoustic startle test	Float

Experimental and technical parameters

Equipment name	Acoustic startle and prepulse inhibition apparatus
Equipment manufacturer	San Diego Instruments
Equipment model	SR-LAB ABS System
Software	SR LAB
Disinfectant	70% ethanol